Antibody targeting compounds

ABSTRACT

The present invention provides antibody targeting compounds in which the specificity of the antibody has been reprogrammed by covalently or noncovalently linking a targeting agent to the combining site of an antibody. By this approach, the covalently modified antibody takes on the binding specificity of the targeting agent. The compound may have biological activity provided by the targeting agent or by a separate biological agent. Various uses of the invention compounds are provided.

BACKGROUND OF THE INVENTION

[0001] The invention relates to compounds for targeting biologicalmolecules and methods of making and using the compounds. Conventionallydeveloped pharmaceutical drugs and biological effector molecules areoften of limited use in therapy because of high toxicity. Variousapproaches have been used over the years to improve the therapeuticindex of such drugs or effectors. One approach has been to couple a drugor effector to a ligand targeting agent such as an antibody. In thiscase, the antibody is used to change the distribution of drug oreffector such that more of it can localize where it is most needed invivo. Improved targeting of small molecular weight drugs or effectorshas been achieved by complexing the drug or effector with a largemolecular weight compound. For example, European Patent EP 217577discloses that increased half life and targeting by an agent is achievedby forming complexes in vivo between hapten-modified agents andanti-hapten antibodies. Similarly, International Patent ApplicationPublication WO 98/22141 discloses conjugates of therapeutic agents andhaptens. The conjugates are administered to a subject and circulate inthe blood stream of the subject. Circulating conjugates are recognizedand bound by existing antibodies in the subject. Also, Shokat andSchultz (J. Am. Chem. Soc., 1991, 113:1862-1864) have disclosed aprocess for redirecting the immune response using a process referred toas ligand-mediated immunogenicity. According to this teaching, aninvariant antigen is complexed with a specific ligand and administeredto a subject. The complexed invariant antigen then binds naturallyoccurring antibodies present in the subject.

BRIEF SUMMARY OF THE INVENTION

[0002] The present invention provides antibody targeting compounds withunique specificity and biological properties which are useful in manyapplications. The antibody targeting compounds of the invention compriseone or more targeting agents or biological agents or both covalently ornoncovalently linked to an antibody combining site. A linear or branchedlinker is preferably used in covalent and non-covalent linkage. Chemicalcharacteristics of the linker are disclosed. Depending on thecircumstances, the antibody specificity of the combining site may bemodified or eliminated following covalent or noncovalent linking to thetargeting or biological agent. In some embodiments, the antigen bindingspecificity of the antibody before covalent linkage may be substantiallyretained after covalent linkage.

[0003] The antibody targeting compound confers various benefits over thecomponents themselves. For example, the antibody portion of the compoundmay generally extend the half-life of a smaller sized targeting orbiological agent in vivo. Also, the biological potency or otherbiological feature of a particular targeting or. biological agent may bemodified by the addition of effector function(s) provided by theantibody portion of the compound (e.g., complement mediated effectorfunctions). In addition, the targeting agent or binding agent, throughits increased size conferred by linkage to the antibody, may enable thetargeting agent to function in new capacities.

[0004] In some embodiments, the targeting agent of the compound can bindto a non-immunoglobulin target molecule or to an immunoglobulin targetmolecule outside of the immunoglobulin combining site. Thus, in theseembodiments, the targeting agent is specific for a non-antibody or isspecific for an antibody but binds to the antibody outside its combiningsite. In a preferred approach, a catalytic antibody can be modified intoa compound that binds specifically to a biomolecule. The antibodyportion of the antibody targeting compounds can include whole antibodyor unique antibody fragments and may have sequence derived from variousanimal species such as a non-human immunoglobulin or humanimmunoglobulin, the latter including a human antibody, humanizedantibody or human chimeric antibody.

[0005] Also provided are methods of producing antibody targetingcompounds of the invention. In one embodiment, an agent-linker compoundcomprising a targeting agent and/or a biological agent is linked to alinker that comprises a reactive group for, covalent reaction with thecombining site of the antibody. In another approach, an antibody-linkercompound is prepared where the linker includes a reactive group forreaction with said one or more targeting agents or biological agents. Inyet another approach, the agents and the antibody can each be linked toa linkers with compatible reactive groups so that the antibody targetingcompound forms when the two linkers covalently bond.

[0006] Further provided are agent-linker compounds comprising atargeting agent, biological agent or both that can be covalently linkedto the combining site of an antibody. In some embodiments, the linkerincludes a reactive group for covalently linking the targeting agent tothe combining site of the antibody. Linkage to the antibody combiningsite may be to a side chain of a reactive amino acid in the combiningsite. In some embodiments, the reactive amino acid is a lysine while thelinker reactive group is a ketone, a diketone, a beta lactam, asuccinimide active ester, haloketone, a lactone, an anhydride, anepoxide, an aldehyde, a halide, a sulfonate, a phosphonate, a guanidine,an amidine, an imine, an eneamine, a ketal, a acetal, or a maleimide.

[0007] Various chemical features of the agent-linker compound aredescribed. In one embodiment, the linker has the general formula X—Y—Zwherein X is a linear or branched connecting chain of atoms comprisingany of C, H, N, O, P, S, Si, F, Cl, Br, and I, or a salt thereof, andcomprising a repeating ether unit of between 2-100 units; Y is optionaland is a single or fused 5 or 6 membered homo- or heterocarbocylicsaturated or unsaturated ring located within 1-20 atoms of Z; and Z is areactive group for covalently linking the one or more targeting agentsto a side chain of a reactive amino acid in the combining site of theantibody. The targeting agent may be linked to X or Y or to X and Y whenmore than one targeting agent or biological agent is included in thetargeting agent-linker compound.

[0008] Yet further provided are targeting agent-linker-antigen compoundsfor noncovalently linking to the combining site of an antibody. Thesecompounds include two or more targeting agents, two or more biologicalagents or at least two agents, one of which is a targeting agent andanother a biological agent. The agents are covalently linked via alinker to an antigen recognized by the antibody. Various chemicalfeatures of the linker and antigen are disclosed.

[0009] Still further provided are methods of modifying an antibody whichexhibits low or nondetectable binding affinity for a particular targetmolecule so that the antibody has increased binding specificity for theparticular target molecule. In one embodiment, one or more targetingagents or biological agents specific for the particular target moleculeare covalently linked to the combining site of the antibody to generatean antibody targeting compound. The agents are linked in such a way asto retain their ability to bind the particular target molecule. In somesuch embodiments, the antibody prior to covalent linking possesses anaffinity for the target molecule of less than about 1×10⁻⁵ moles/liter.After covalent linking, the targeting compound may exhibit an affinityfor the target molecule of greater than about 1×10⁻⁶ moles/liter.

[0010] Additionally provided are methods of altering at least onephysical or biological characteristic of a targeting agent or biologicalagent. In one embodiment, the agent is covalently linked to thecombining site of an antibody to generate an antibody targetingcompound. Methods are also provided for modifying one or more physicalor biological properties of the antibody targeting compounds bymodifying one or more chemical characteristics of the linker. In someembodiments, the physical or biological properties modified includepharmacokinetics, pharmacodynamics, immunogenicity, binding affinity,susceptibility to degradation, solubility, lipophilicity,hydrophilicity, hydrophobicity, stability, and rigidity.

[0011] Also provided are methods of delivering a biological activity tocells, an extracellular matrix biomolecule or a fluid biomolecule of anindividual. In one approach an antigen targeting compound of theinvention which is biologically active and is specific for the cells,extracellular matrix biomolecule or fluid biomolecule is administered tothe individual. In another approach, an agent-linker-antigen compound ofthe invention, specific for cells, tissue extracellular matrixbiomolecule or fluid biomolecule, and an antibody specific for theantigen are separately administered to the individual and the antibodytargeting agent forms in vivo when the agent-linker-antigen compoundnon-covalently associates with the antibody combining site.

[0012] Further provided are methods treating or preventing a disease orcondition in an individual wherein the disease or condition involvescells, tissue or fluid that expresses a target molecule. In oneapproach, a therapeutically effective amount of an antibody targetingcompound of the invention is administered to the individual. In anotherapproach, a therapeutically effective amount of an agent-linker-antigencompound of the invention, and an antibody specific for the antigen areseparately administered to the individual and the antibody targetingagent forms in vivo when the agent-linker-antigen compoundnon-covalently associates with the antibody combining site. In bothapproaches, the antibody targeting compound or agent-linker-antigencompound is specific for the target molecule, and the compound orantibody comprises a biological activity effective against the diseaseor condition.

[0013] Still further provided are methods of imaging cells orextracellular matrix in an individual wherein the cells or extracellularmatrix express a target molecule. In one approach, an antibody targetingcompound of the invention is linked to a detectable label andadministered to the individual. In another approach anagent-linker-antigen compound and an antibody specific for the antigenare separately administered to the individual and the antibody targetingagent forms in vivo when the agent-linker-antigen compoundnon-covalently associates with the antibody combining site. In bothapproaches, the label may be linked to the antibody, the targeting agentand/or biological agent.

[0014] Additionally provided are methods of reducing the infectivity ofmicrobial cells or viral particles present on a surface. According tothese methods, the surface is contacted with an effective amount of anantibody targeting compound of the invention, wherein the antibodytargeting compound comprises a targeting agent or biological agentspecific for a receptor on said microbial cells or virus particles.

[0015] Also provided are methods of screening a chemical library foragonists or antagonists of a receptor. The method includes linkingindividual members of the chemical library to the combining site of anantibody and then testing the antibody linked library for binding to thereceptor or for inhibition of binding between the receptor and a ligandfor the receptor.

[0016] Further provided are various immunoassays that use antibodytargeting compounds of the invention. In one embodiment for detecting ormeasuring analyte in a sample, the invention comprises use of anantibody targeting compound of the invention wherein the antibodyspecificity for the analyte results from the targeting agent, which iscovalently linked to the antibody combining site. In another embodimentinvolving a direct or indirect binding assay for determining thepresence of an analyte using an antibody specific for the analyte, theinvention comprises determining the presence of the analyte using anantibody specific for the analyte wherein the antibody specificityresults from a non-antibody targeting agent specific for the analytethat is linked to a reactive amino acid in the combining site of theantibody.

[0017] Still further provided are methods of inhibiting or reducing theability of a targeting agent or biological agent to cross a cellmembrane. In these methods an antibody targeting compound is formed bycovalently linking the combining site of an antibody that does notitself cross the cell membrane to the targeting agent or biologicalagent, wherein linkage of said antibody to said targeting agent orbiological agent reduces or inhibits the ability of the agent to crossthe cell membrane.

[0018] Additionally provided are methods of mediating intracellulardelivery of a intracellularly active drug. In these methods, an antibodytargeting compound is prepared wherein said compound includes one ormore targeting agents or one or more biological agents or bothcovalently linked via a linker to the combining site of the antibody.The targeting agents or biological agents are characterized in that theybind to a cell receptor and mediate internalization of the agent. Theantibody targeting compound also includes a drug that is activeintracellularly. Intracellular drug delivery occurs when a cellexpressing the receptor contacts the antibody targeting compound. Thecontacting results in internalization of the antibody targeting agentand delivery of said drug intracellularly. In some embodiments, theintracellularly active drug is a prodrug that becomes active when saiddrug contacts an intracellular compartment. The antibody targetingcompound may include an intracellular trafficking signal to direct theinternalized antibody targeting compound to a particular intracellularcompartment.

[0019] The invention further provides pharmaceutical compositions ormedicaments that include an antibody targeting compound of the inventionand a pharmaceutically acceptable carrier.

BRIEF DESCRIPTION OF THE DRAWINGS

[0020]FIG. 1 shows exemplary integrin targeting agents of which PanelsA-E are RGD peptidomimetic while Panel F is an RGD peptide. The corestructures are from the following: U.S. Pat. Nos. 6,335,330 (Panel A),5,693,636 (Panel B), 6,040,311 (Panel C), and 6,001,117 (Panel E).

[0021]FIG. 2 shows a general scheme of a targeting agent-linker compoundwith a non-branched linker (Panel A) with specific embodiments in PanelB (SCS-873), Panel C (PST inhibitor diketo linker; compound 26), Panel D(TAK-799 diketo linker; compound 27) and Panel E (folate ligand dikonelinker; compound 28).

[0022]FIG. 3 shows a general scheme of an embodiment of a targetingagent-linker compound with a branched linker and two identical targetingagents (Panel A) with specific embodiments in Panel B (integrintargeting agent diketo linker; compound 29), and Panel C (integrintargeting agent diketo linker; compound 30). The branch point is in theconnecting chain portion of the linker.

[0023]FIG. 4 shows a general scheme of an embodiment of a targetingagent-linker compound with a branched linker and two different targetingagents (Panel A) with a specific embodiment in Panel B (integrintargeting and folate targeting agent diketo linker; compound 31). Thebranch point is in the connecting chain portion of the linker.

[0024]FIG. 5 shows a general scheme of an embodiment of a targetingagent-linker compound with a branched linker and two different targetingagents (Panel A) with a specific embodiment in Panel B (integrintargeting agent diketo linker; compound 32). The branch point is in therecognition group portion of the linker.

[0025]FIG. 6 shows the structure of linker reactive groups. StructuresA-C form reversible covalent bonds with reactive nucleophilic group(e.g. lysine or cysteine side chain) in the combining site of anantibody (structure A could form an irreversible covalent bond X is Nand if R₁ and R₃ form part of a cyclic structure). R₁ and R₂ and R₃ instructures A-C represent substituents which can be C, H, N, O, P, S, Si,halogen (F, Cl, Br, I) or a salt thereof. X is N, C, Si, or any otherheteroatom. These substituents may also include a group such as analkyl, alkenyl, alkynyl, oxoalkyl, oxoalkenyl, oxoalkynyl, aminoalkyl,aminoalkenyl, aminoalkynyl, sulfoalkyl, sulfoalkenyl, or sulfoalkynylgroup, phosphoalkyl, phosphoalkenyl, phosphoalkynyl group. R₂ and R₃could be cyclic as exemplified in structures B and C while X could be aheteroatom. Structures D-G form nonreversible covalent bonds withreactive nucleophilic group (e.g. lysine or cysteine side chain) in thecombining site of an antibody. In these structures, R₁ and R₂ representC, O N, halide and leaving groups such as mesyl or tosyl.

[0026]FIG. 7 shows various electrophiles suitable for reactivemodification with a reactive amino acid side chain of an antibody. Key:(A) acyl beta-lactam; (B) simple diketone; (C) succinimide active ester;(D) maleimide; (E) haloacetamide with linker; (F) haloketone; (G)cyclohexyl diketone; and (H) aldehyde. R refers to other structure thatmay include a targeting agent, linker or antibody, while X refers tohalogen.

[0027]FIG. 8 shows the structure of linker recognition group (Y),situated between the reactive group portion and the connecting chainportion of the linker. Panel A shows the relationship of the recognitiongroup Y within the linker (see FIG. 2). Panels B-D show distance of Yfrom Z, substituents on the ring and ring member atoms.

[0028]FIG. 9 shows the structure of the linker connecting chain (X),which directly attaches at one end to the targeting agent as shown inPanel A (see FIG. 2). Substituents R₂ to R₄ are C, H, N, O, P, S, Si,halogen (F, Cl, Br, I) or a salt thereof, and may include a group suchas an alkyl, alkenyl, alkynyl, oxoalkyl, oxoalkenyl, oxoalkynyl,aminoalkyl, aminoalkenyl, aminoalkynyl, sulfoalkyl, sulfoalkenyl,sulfoalkynyl group, phosphoalkyl, phosphoalkenyl, phosphoalkynyl as wellas a carbocyclic or heterocyclic mono or fused saturated or unsaturatedring structure. Panel B: R1 is O and R2 is C, H, N, O, P, S, Si, halogen(F, Cl, Br, I) or a salt thereof. In the connecting chain in structuresB and C, n, r or m is 1-100. In structures D and E, n is 1, 2, 4, ormore preferably is 3.

[0029]FIG. 10 shows Scheme 1, a synthetic scheme for the amine precursorof SCS-873, targeting agent 3 or SCS-amine. Key: (a) BBr₃, CH₂Cl₂, −20°C., 2 h; (b) DMF, rt to 80° C., 3 h; (c) BnCOCl, sat. aq. NaHCO₃, ether;(d) TBDPSiCl, imidazole, DMF, 16 h; (e) Pd(OAc)₂, (o-tol)₃P, i-Pr₂EtN,CH₃CH₂CN, reflux, 3 h; (f) 20 % (w/w) Pd-C (10%), H₂, EtOH-AcOH (1:1),36 h; (g) TBAF, THF, rt, 1 h; (h) DEAD, PPh₃, THF-benzene (3:1), 16 h;(i) 20 % (w/w) Pd-C (10%), cyclohexene-i-PrOH (1:1), 90° C., 12 h; (j)i. aq. 2N NaOH, MeOH-THF (1:1), 16 h, ii. TFAA, anisole, CH₂Cl₂, 0° C.,2 h.

[0030]FIG. 11 shows Scheme 2, a synthetic scheme for making Compound 4,(R=Butoxycarboxyaminohexanoyl-derivative). Key: (a) DMF, rt; (b) EDC,HOBT, DMF; (c) 0.01 M in DMSO, 130° C.; (d) TFAA, anisole,dichloromethane; (e) DMF; (f) EDC, HOBT, DMF; (g) (i) step d, (ii) 2MNaOH, MeOH-THF (1:1).

[0031]FIG. 12 shows Scheme 3, a synthetic scheme for making compoundsSCS-873 and SCS-1655.

[0032]FIG. 13 shows Scheme 4, a synthetic scheme for making CompoundsSCS-864 and SCS-789. Key: (a) Et₃N, DMF, rt, 16 h.

[0033]FIG. 14 shows a scheme for forming a targeting agent-linkercompound using a linker with a maleimide-diketone reactive group.

DETAILED DESCRIPTION OF THE INVENTION

[0034] The present invention provides various antibody targetingcompounds in which targeting agents and/or biological agents arecovalently or noncovalently linked to the combining site of an antibody.When one or more targeting agents are linked, at least one of thetargeting agents will be linked so that it can bind its target. This maybe achieved by linking the targeting agent in a manner that does effectits binding specificity for the target and by sufficiently distancingthe targeting agent from the antibody combining site so that it can bindits target without steric hindrance by the antibody. This may beachieved by using a suitable linker and linking strategy discussed inmore detail ahead.

[0035] When a biological agent is not also a targeting agent it ispreferred that the antibody retain at least some antigen bindingspecificity following linkage to one or more biological agents. Theantibody compound in which one or more biological agents are linked tothe antibody combining site may exhibit biological activity due to alinked biological agent if such agent is biologically active whilelinked to the antibody. This may be achieved by various strategies suchas by linking the antibody combining site to a location on thebiological agent that does not affect biological activity. Anotherstrategy is to position the biological agent away from the antibody sothat the biological agent can bind to another molecule necessary foractivity without steric hindrance by the antibody. Other strategies forobtaining a biological activity of one or more biological agents linkedto the antibody combining site are well known to the skilled artisan. Insome embodiments, the biological activity of a biological agent may notbe realized until the agent is released from the antibody combiningsite. This may be achieved in some embodiments though the aid of labilelinkage as discussed further ahead.

[0036] In some embodiments, the native antigen binding specificity ofthe antibody which exists before covalent linkage will not besubstantially modified following covalent linkage. In other words, theantibody compound resulting from covalent linkage of one or moretargeting agents or one or more biological agents may bind the sameantigens with a similar affinity as it did prior to covalent linkage. Inother embodiments, the binding specificity of the antibody beforecovalent linkage will be substantially modified following covalentlinkage. Substantially modified antibody binding specificity resultingfrom covalent linkage may be due to a substantially reduced ability ofthe covalently linked antibody to bind to an antigen or a substantiallyincreased ability of the covalently linked antibody to bind to anantigen. In some embodiments, binding of the antigen binding site toantigen is sufficiently reduced such that the original antigen bindingspecificity of the antibody is effectively eliminated. In someembodiments, the antigen binding site to antigen is sufficiently reducedsuch that the original antigen binding specificity of the antibody iseffectively eliminated and replaced with that of a targeting agent(s)covalently linked to the antibody combining site. In embodiments wherethe binding specificity of the antibody is effectively replaced withthat of the targeting agent(s), the antibody, after covalent linkage tothe targeting agent(s), exhibits an affinity for the target molecule ofgreater than about 1×10⁻⁶ moles/liter.

[0037] Although not wishing to be bound by any theory, substantiallyreduced antibody binding to antigen may result from the targetingagent(s) or biological agent(s) sterically hindering the antigen fromcontacting the antibody combining site. Alternatively, or in addition,substantially reduced antigen binding may result if the amino acid sidechain of the antibody combining site modified by covalent linkage wasimportant for binding to the antigen. Substantially increased antibodybinding to an antigen may result when the targeting agent(s) orbiological agent(s) do not sterically hinder the antigen from contactingthe antibody combining site and amino acid side chain of the antibodycombining site modified by covalent linkage was important for binding tothe antigen.

[0038] The targeting compounds of the invention may comprise an antibodyor an antibody fragment that has a single combining site such as Fab orFab′ antibody fragments. In such cases, the targeting agent will belinked to the single combining site of that antibody molecule. If anantibody or antibody fragment of a targeting molecule comprises two ormore combining sites, at least one of the combining sites will include acovalently linked targeting agent. In some cases, all or most of thecombining sites of an antibody can be covalently linked to a targetingagent. If multiple combining sites of an antibody are to be linked totargeting agents, the combining sites may all have the same targetingagent linked thereto or may have different targeting agents linked tothe same antibody. It would be readily understood that one couldcovalently link multiple targeting agents to a single antibody combiningsite. Such multimeric targeting agents may be heteromultimeric orhomomultimeric with respect to the specificity of the targeting agentsin the multimer.

[0039] “Targeting agent” or “targeting component” as used herein refersto a moiety that recognizes, binds or adheres to a target moiety of atarget molecule located for example in a cell, tissue (e.g.extracellular matrix), fluid, organism, or subset thereof. A targetingagent and its target molecule represent a binding pair of molecules,which interact with each other through any of a variety of molecularforces including, for example, ionic, covalent, hydrophobic, van derWaals, and hydrogen bonding, so that the pair have the property ofbinding specifically to each other. Specific binding means that thebinding pair exhibit binding with each other under conditions where theydo not bind to another molecule. Examples of binding pairs arebiotin-avidin, hormone-receptor, receptor-ligand, enzyme-substrate,IgG-protein A, antigen-antibody, and the like. The targeting agent andits cognate target molecule exhibit a significant association for eachother. This association may be evaluated by determining an equilibriumassociation constant (or binding constant) according to methods wellknown in the art. Affinity is calculated as K_(d)=k_(off)/k_(on)(k_(off) is the dissociation rate constant, k_(on) is the associationrate constant and K_(d) is the equilibrium constant.

[0040] Affinity can be determined at equilibrium by measuring thefraction bound (r) of labeled ligand at various concentrations (c). Thedata are graphed using the Scatchard equation: r/c=K(n−r):

[0041] where

[0042] r=moles of bound ligand/mole of receptor at equilibrium;

[0043] c=free ligand concentration at equilibrium;

[0044] K=equilibrium association constant; and

[0045] n=number of ligand binding sites per receptor molecule

[0046] By graphical analysis, r/c is plotted on the Y-axis versus r onthe X-axis thus producing a Scatchard plot. The affinity is the negativeslope of the line. k_(off) can be determined by competing bound labeledligand with unlabeled excess ligand (see, e.g., U.S. Pat. No.6,316,409). The affinity of a targeting agent for its target molecule ispreferably at least about 1×10⁻⁶ moles/liter, is more preferably atleast about 1×10⁻⁷ moles/liter, is even more preferably at least about1×10⁻⁸ moles/liter, is yet even more preferably at least about 1×10⁻⁹moles/liter, and is most preferably at least about 1×10⁻¹⁰ moles/liter.

[0047] Targeting agents include, but are not limited to, small moleculeorganic compounds of 5,000 daltons or less such as drugs, proteins,peptides, peptidomimetics, glycoproteins, proteoglycans, lipidsglycolipids, phospholipids, lipopolysaccharide, nucleic acids,proteoglycans, carbohydrates, and the like. Targeting agents may includewell known therapeutic compounds including anti-neoplastic agents.Anti-neoplastic targeting agents may include targpaclitaxel,daunorubicin, doxorubicin, carminomycin, 4′-epiadriamycin,4-demethoxy-daunomycin, 11-deoxydaunorubicin, 13-deoxydaunorubicin,adriamycin-14-benzoate, adriamycin-14-octanoate,adriamycin-14-naphthaleneacetate, vinblastine, vincristine, mitomycin C,N-methyl mitomycin C, bleomycin A₂, dideazatetrahydrofolic acid,aminopterin, methotrexate, cholchicine and cisplatin, and the like.Anti-microbial agents include aminoglycosides including gentamicin,antiviral compounds such as rifampicin, 3′-azido-3′-deoxythymidine (AZT)and acylovir, antifungal agents such as azoles including fluconazole,plyre macrolides such as amphotericin B, and candicidin, anti-parasiticcompounds such as antimonials, and the like. Hormone targeting agentsinclude toxins such as diphtheria toxin, cytokines such as CSF, GSF,GMCSF, TNF, erythropoietin, immunomodulators or cytokines such as theinterferons or interleukins, a neuropeptide, reproductive hormone suchas HGH, FSH, or LH, thyroid hormone, neurotransmitters such asacetylcholine, and hormone receptors such as the estrogen receptor.

[0048] In some preferred embodiments, the targeting agent is not anantibody. In other preferred embodiments, the targeting agent is not ametal chelate. Preferably, the targeting agent is a small molecule ascompared with a native immunoglobulin. The targeting agent, includingany linking moiety necessary for covalently linking the targeting agentto an amino acid residue of the antibody combining site, preferably isat least about 300 daltons in size, and preferably may be at least about400, 500, 600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500,1,600, 1,700, 1,800, 1,900, 2,000, 2,500, 3,000, 3,500, 4,000, 4,500 oreven 5,000 daltons in size, with even larger sizes possible.

[0049] Suitable targeting agents in targeting compounds of the inventioncan be a protein or peptide. “Polypeptide”, “peptide,” and “protein” areused interchangeably to refer to a polymer of amino acid residues. Asused herein, these terms apply to amino acid polymers in which one ormore amino acid residue is an artificial chemical analogue of acorresponding naturally occurring amino acid. These terms also apply tonaturally occurring amino acid polymers. Amino acids can be in the L orD form as long as the binding function of the peptide is maintained.Peptides can be of variable length, but are generally between about 4and 200 amino acids in length. Peptides may be cyclic, having anintramolecular bond between two non-adjacent amino acids within thepeptide, e.g., backbone to backbone, side-chain to backbone andside-chain to side-chain cyclization. Cyclic peptides can be prepared bymethods well know in the art. See e.g., U.S. Pat. No. 6,013,625.

[0050] Protein or peptide targeting agents that exhibit binding activityfor a target molecule are well known in the art. For example, atargeting agent may be a viral peptide cell fusion inhibitor. This mayinclude the T-20 HIV-1 gp41 fusion inhibitor which targets fusionreceptors on HIV infected cells (for T-20, see U.S. Pat. Nos. 6,281,331and 6,015,881 to Kang et al.; Nagashima et al. J. Infectious Diseases183:1121, 2001; for other HIV inhibitors see U.S. Pat. No. 6,020,459 toBarney and WO 0151673A2 to Jeffs et al), RSV cell fusion inhibitors (seeWO 0164013A2 to Antczak and McKimm-Breschkin, Curr. Opin. Invest. Drugs1:425-427, 2000 (VP-14637)), pneumovirus genus cell fusion inhibitors(see WO 9938508A1 by Nitz et al.), and the like. Targeting agents alsoinclude peptide hormones or peptide hormone analogues such as LHRH,bombesin/gastrin releasing peptide, somatastatin (e.g., RC-121octapeptide), and the like, which may be used to target any of a varietyof cancers ovarian, mammary, prostate small cell of the lung,colorectal, gastric, and pancreatic. See, e.g., Schally et al., Eur. J.Endocrinology, 141:1-14, 1999.

[0051] Peptide targeting agents suitable for use in targeting compoundsof the invention also may be identified using in vivo targeting of phagelibraries that display a random library of peptide sequences (see, e.g.,Arap et al., Nature Medicine, 2002 8(2):121-7; Arap et al., Proc. Natl.Acad. Sci. USA 2002 99(3):1527-1531; Trepel et al. Curr. Opin. Chem.Biol. 2002 6(3):399-404).

[0052] In some embodiments, the targeting agent is specific for anintegrin. Integrins are heterodimeric transmembrane glycoproteincomplexes that function in cellular adhesion events and signaltransduction processes. Integrin α_(v)β₃ is expressed on numerous cellsand has been shown to mediate several biologically relevant processes,including adhesion of osteoclasts to bone matrix, migration of vascularsmooth muscle cells, and angiogenesis. Integrin α_(v)β₃ antagonistslikely have use in the treatment of several human diseases, includingdiseases involving neovascularization, such as rheumatoid arthritis,cancer, and ocular diseases.

[0053] Suitable targeting agents for integrins include RGD peptides orpeptidomimetics or non-RGD peptides or peptidomimetics. As used herein,reference to “Arg-Gly-Asp peptide” or “RGD peptide” is intended to referto a peptide having one or more Arg-Gly-Asp containing sequence whichmay function as a binding site for a receptor of the “Arg-Gly-Asp familyof receptors”, e.g., an integrin. Integrins, which comprise and alphaand a beta subunit, include numerous types including α₁β₁, α₂β₁, α₃β₁,α₄β₁, α₅β₁, α₆β₁, α₇β₁, α₈β₁, α₉β₁, α₁β₁, α₆β₄, α₄β₇, α_(D)β₂, α_(D)β₂,α_(L)β₂, α_(M)β₂, α_(v)β₁, α_(v)β₃, α_(v)β₅, α_(v)β₆, α_(v)β₈, α_(x)β₂,α_(IIb)β₃, α_(IELb)β₇, and the like. The sequence RGD is present inseveral matrix proteins and is the target for cell binding to matrix byintegrins. Platelets contain a large amount of RGD-cell surfacereceptors of the protein GP II_(b)/III_(a), which is primarilyresponsible, through interaction with other platelets and with theendothelial surface of injured blood vessels, for the development ofcoronary artery thrombosis. The term RGD peptide also includes aminoacids that are functional equivalents (e.g., RLD or KGD) thereofprovided they interact with the same RGD receptor. Peptides containingRGD sequences can be synthesized from amino acids by means well known inthe art, using, for example, an automated peptide synthesizer, such asthose manufactured by Applied Biosystems,Inc., Foster City, Calif.

[0054] As used herein, “non-RGD” peptide refers to a peptide that is anantagonist or agonist of integrin binding to its ligand (e.g.fibronectin, vitronectin, laminin, collagen etc.) but does not involvean RGD binding site. Non-RGD integrin peptides are known for α_(v)β₃(see, e.g., U.S. Pat. Nos. 5,767,071 and 5,780,426) as well as for otherintegrins such as α₄β₁ (VLA-4), α₄β₇ (see, e.g., U.S. Pat. No.6,365,619; Chang et al., Bioorganic & Medicinal Chem Lett, 12:159-163(2002); Lin et al., Bioorganic & Medicinal Chem Lett, 12:133-136(2002)), and the like.

[0055] An integrin targeting agent may be a peptidomimetic agonist orantagonist, which preferably is a peptidomimetic agonist or antagonistof an RGD peptide or non-RGD peptide. As used herein, the term“peptidomimetic” is a compound containing non-peptidic structuralelements that are capable of mimicking or antagonizing the biologicalaction(s) of a natural parent peptide. A peptidomimetic of an RGDpeptide is an organic molecule that retains similar peptide chainpharmacophore groups of the RGD amino acid sequence but lacks aminoacids or peptide bonds in the binding site sequence. Likewise, apeptidomimetic of a non-RGD peptide is an organic molecule that retainssimilar peptide chain pharmacophore groups of the non-RGD binding sitesequence but lacks amino acids or peptide bonds in the binding sitesequence. A “pharmacophore” is a particular three-dimensionalarrangement of functional groups that are required for a compound toproduce a particular response or have a desired activity. The term “RGDpeptidomimetic” is intended to refer to a compound that comprises amolecule containing the RGD pharmacophores supported by anorganic/non-peptide structure. It is understood that an RGDpeptidomimetic (or non-RGD peptidomimetic) may be part of a largermolecule that itself includes conventional or modified amino acidslinked by peptide bonds.

[0056] RGD peptidomimetics are well known in the art, and have beendescribed with respect to integrins such as GPIIb/IIIa, α_(v)β₃ andα_(v)β₅ (See, e.g., Miller et al., J. Med. Chem. 2000, 43:22-26; andInternational Pat. Publications WO 0110867, WO 9915178, WO 9915170, WO9815278, WO 9814192, WO 0035887, WO 9906049, WO 9724119 and WO 9600730;see also Kumar et al., Cancer Res. 61:2232-2238 (2000)). Many suchcompounds are specific for more than one integrin. RGD peptidomimeticsare generally based on a core or template (also referred to as“fibrinogen receptor antagonist template”), to which are linked by wayof spacers to an acidic group at one end and a basic group at the otherend of the core. The acidic group is generally a carboxylic acidfunctionality while the basic group is generally a N-containing moietysuch as an amidine or guanidine. Typically, the core structure adds aform of rigid spacing between the acidic moiety and the basic nitrogenmoiety, and contains one or more ring structures (e.g., pyridine,indazole, etc.) or amide bonds for this purpose. For a fibrinogenreceptor antagonist, generally, about twelve to fifteen, more preferablythirteen or fourteen, intervening covalent bonds are present (via theshortest intramolecular path) between the acidic group of the RGDpeptidomimetic and a nitrogen of the basic group. The number ofintervening covalent bonds between the acidic and basic moiety isgenerally shorter, two to five, preferably three or four, for avitronectin receptor antagonist. The particular core may be chosen toobtain the proper spacing between the acidic moiety of the fibrinogenantagonist template and the nitrogen atom of the pyridine. Generally, afibrinogen antagonist will have an intramolecular distance of about 16angstroms (1.6 nm) between the acidic moiety (e.g., the atom which givesup the proton or accepts the electron pair) and the basic moiety (e.g.,which accepts a proton or donates an electron pair), while a vitronectinantagonist will have about 14 angstroms (1.4 nm) between the respectiveacidic and basic centers. Further description for converting from afibrinogen receptor mimetic to a vitronectin receptor mimetic can befound in U.S. Pat. No. 6,159,964.

[0057] The peptidomimetic RGD core can comprise a 5-11 membered aromaticor nonaromatic mono- or polycyclic ring system containing 0 to 6 doublebonds, and containing 0 to 6 heteroatoms chosen from N, O and S. Thering system may be unsubstituted or may be substituted on a carbon ornitrogen atom. Preferred core structures with suitable substituentsuseful for vitronectin binding include monocyclic and bicyclic groups,such as benzazapine described in WO 98/14192, benzdiazapine described inU.S. Pat. No. 6,239,168, and fused tricyclics described in U.S. Pat No.6,008,213.

[0058] U.S. Pat. No. 6,159,964 contains an extensive list of referencesin Table 1 of that document which disclose RGD peptidomimetic coresstructures (referred to as fibrinogen templates) which can be used forprepraring RGD peptidomimetics. Preferred vitronectin RGD andfibronectin RGD peptidomimetics are disclosed in U.S. Pat. Nos.6,335,330; 5,977,101; 6,088,213; 6,069,158; 6,191,304; 6,239,138;6,159,964; 6,117,910; 6,117,866; 6,008,214; 6,127,359; 5,939,412;5,693,636; 6,403,578; 6,387,895; 6,268,378; 6,218,387; 6,207,663;6,011,045; 5,990,145; 6,399,620; 6,322,770; 6,017,925; 5,981,546;5,952,341; 6,413,955; 6,340,679; 6,313,119; 6,268,378; 6,211,184;6,066,648; 5,843,906; 6,251,944; 5,952,381; 5,852,210; 5,811,441;6,114,328; 5,849,736; 5,446,056; 5,756,441; 6,028,087; 6,037,343;5,795,893; 5,726,192; 5,741,804; 5,470,849; 6,319,937; 6,172,256;5,773,644; 6,028,223; 6,232, 308; 6,322,770; 5,760,028.

[0059] Exemplary RGD peptidomimetic integrin targeting agents are shownbelow as compounds 1, 2, and 3 can be used for preparing an intregrintargeting compound of the present invention. In the three compounds, thelinker is attached as indicated to the nitrogen of the seven memberedring. Other RGD peptidomimetic integrin targeting agents includecompound 33, wherein P and L or carbon or nitrogen. The linker may be R1or R2 while the R3 group includes a basic group such as an —NH group. Insome embodiments, the R3 group is as shown in compounds 1, 2, or 33. Insome embodiments, the R3 group includes a heterocyclic group such abenzimidazole, imidazole, pyridine group, or the like. In some suchembodiments, the R3 group is a alkoxy group, such as a propoxy group orthe like, that is substituted with a heterocyclyl group that issubstituted with an alkylamine group, such as a methylamino group or thelike, whereas in other embodiments, the R3 group is an alkoxy group,such as a propoxy group or the like, substituted with aheterocyclylamino group, such as with a pyridinylamino group or the likesuch as a 2-pyridinylamino group. In other embodiments R3 is a group offormula —C(═O)Rb where Rb is selected from —N(alkyl)-alkyl-heterocyclylgroups such as —N(Me)—CH2-benzimidazole groups and the like.

[0060] Other exemplary integrin peptidomimetic targeting agents and apeptide targeting agent are shown in FIG. 1. The linker may be any ofR₁, R₂, R₃, while R₄ may be a linker or a hydrolyzable group such asalkyl, alkenyl, alkynyl, oxoalkyl, oxoalkenyl, oxoalkynyl, aminoalkyl,aminoalkenyl, aminoalkynyl, sulfoalkyl, sulfoalkenyl, or sulfoalkynylgroup, phosphoalkyl, phosphoalkenyl, phosphoalkynyl group, and the like.One of skill in the art will readily appreciate that other integrinagonist and antagonist mimetics can also be used in targeting compoundsof the present invention.

[0061] The target molecule to which the targeting agent of the targetingcompound binds is preferably a non-immunoglobulin molecule or is animmunoglobulin molecule where the target moiety is outside theimmunoglobulin combining site. It is not intended to exclude from theinventive compounds those targeting agents that function as antigensand, therefore, bind to an immunoglobulin combining site. Such targetingagents are included herein provided the targeting agents also bind to anon-immunoglobulin molecule and/or a target moiety located outside thecombining site of an immunoglobulin molecule. In general, the targetmolecule can be any type of molecule including organic, inorganic,protein, lipid, carbohydrate, nucleic acid and the like.

[0062] The target molecule is preferably a biomolecule such as aprotein, carbohydrate, lipid or nucleic acid. The target molecule can beassociated with a cell (“cell surface expressed”), or other particle(“particle surface expressed”) such as a virus, or may be extracellular.If associated with a cell or particle, the target molecule is preferablyexpressed on the surface of the cell or particle in a manner that allowsthe targeting agent of the targeting compound to make contact with thesurface receptor from the fluid phase of the body.

[0063] In some preferred embodiments, the target molecule ispredominantly or exclusively associated with a pathological condition ordiseased cell, tissue or fluid. Thus, the targeting agent of a presentantibody targeting compound can be used to deliver the targetingcompound to a diseased tissue by targeting the cell, an extracellularmatrix biomolecule or a fluid biomolecule. Exemplary target moleculesdisclosed hereinafter in the Examples include integrins (Example 1),cytokine receptors (Examples 2, 3 and 7), cytokines (Example 4), vitaminreceptors (Example 5), cell surface enzymes (Example 6), and HIV-1 virusand HIV-1 virus infected cells (Examples 8 and 11), and the like.

[0064] In other preferred embodiments, the target molecule is associatedwith an infectious agent and is expressed on the surface of a microbialcell or on the surface of a viral particle. As such, antibody targetingcompositions in which the targeting agent can bind to the cell surfaceexpressed or particle expressed infectious agent can be used as ananti-microbial, by targeting microbial agents inside the body or on thesurface (e.g., skin) of an individual. In the latter case, the inventioncompound can be applied topically.

[0065] Antibody targeting agents specific for a microbial targetmolecule also can be used as an anti-microbial agent in vitro.Accordingly, a method of reducing the infectivity of microbial cells orviral particles present on a surface is provided. Some methods includecontacting the surface of a microbial cell or viral particle with aneffective amount of the invention targeting compound. The targetingcompound in such methods includes a targeting agent specific for areceptor on the microbial cell or virus particle. Applicable surfacesare any surfaces in vitro such as a counter top, condom, and the like.

[0066] Another preferred target molecule for targeting molecules of theinvention is prostate specific antigen (PSA), a serine protease that hasbeen implicated in a variety of disease states including prostatecancer, breast cancer and bone metastasis. Specific inhibitors of PSAwhich bind to the active site of PSA are known. See Adlington et al., J.Med. Chem., 2001, 44:1491-1508 and WO 98/25895 to Anderson. A specificinhibitor of PST is shown below as compound 34.

[0067] A targeting agent, in addition to its ability to bind a targetmolecule, may be characterized in having one or more biologicalactivities, each activity characterized as a detectable biologicalaffect on the functioning of a cell organ or organism. Thus, in additionto being a targeting agent, such compounds can be considered biologicalagents. For example, the integrin targeting agents shown as compounds 1,2, 3 and 33 above not only target an integrin, but have integrinantagonist biological activity. In some embodiments, however, atargeting agent may be a pure binding agent without biological activity.

[0068] The targeting compounds of the invention include a targetingagent that is covalently linked to a combining site of an antibody. Suchtargeting compounds may have one or more biological activitiesassociated with the targeting compound. The biological activity may bean inherent feature of the targeting agent itself or may be provided bya biological agent distinct from the targeting agent in the targetingcompound. The biological agent may be associated covalently ornon-covalently with the other molecules or portions of the targetingcompound, although covalent linkage is preferred. The biological agentmay be linked to either the targeting agent, the antibody, or both bymeans well known in the art. For example, see Kiaris et al., Eur. J.Cancer 37:620-628 (2001) and Schally et al. Eur. J. Endocrin. 141:1-14(1989), which describe various conjugates between peptide hormonetargeting agents and doxorubicin. See also, Canevari et al., Ann Oncol1994 October;5(8):698-701; Rihova, Folia Microbiol (Praha)1995;40(4):367-84; Vitetta, Princess Takamatsu Symp 1988;19:333-40; andGhose et al., Crit Rev Ther Drug Carrier Syst 1987;3(4):263-359. Thus,in some embodiments, the antibody-targeting agent targeting compounds ofthe invention may include a functional component in the form of atargeting agent that has inherent biological activity. In suchembodiments, the targeting agent is linked to a combining site of theantibody or antibody fragment and the targeting agent is the functionalcomponent that exhibits the biological activity. In other embodiments,the targeting compound includes a targeting agent linked to a combiningsite of an antibody or antibody fragment, and also includes a separatefunctional component that is preferably attached or linked to thetargeting compound through a covalent bond.

[0069] A targeting agent or biological agent can be linked to anantibody targeting compound of the invention using a linkage that islabile under certain conditions. The labile linkage may be between theantibody and the targeting agent or biological agent, while if a linkeris present, the labile linkage may be between the antibody and thelinker, the targeting agent or biological agent and the linker, withinthe linker, or combinations thereof.

[0070] Labile linkers include, reversible covalent bonds, pH sensitivelinkages (acid or base sensitive), enzyme sensitive linkages,degradation sensitive linkers, photosensitive linkers, sand the like,and combinations thereof. These features are also characteristic of aprodrug which can be considered as a type of labile linker. A variety oflabile linkers have been previously designed. For example, prodrugs canbe formed using compounds having carboxylic acid moieties that slowlydegrade by hydrolysis as described in U.S. Pat. No. 5,498,729.

[0071] The particular design of a labile linker may be used to directrelease of the biological agent after it has reached the intendedtarget. For example, a linkage may be designed to direct release in aparticular intracellular compartment or in an extracellular compartmentin which antibody targeting compounds may accumulate. An acid-labilelinker such as a cis-aconitic acid linker can take advantage of theacidic environment of different intracellular compartments such as theendosomes encountered during receptor mediated endocytosis and thelysosomes. See Shen et al., Biochem. Biophys. Res. Commun. (1981)102:1048-1054; Yang et al., J. Natl. Canc. Inst. (1988) 80: 1154-1159. Apeptide spacer arm located within or at the ends of a linker can be usedto effect release of a targeting agent or biological agent by the actionof a peptidase such as a lysosomal peptidase. See e.g., Trouet et al.,Proc. Natl. Acad. Sci. (1982) 79: 626-629.

[0072] Particular targeting agents may or may not possess biologicalactivity depending on the context of their use. For example, thetherapeutic drug doxorubicin, which is a DNA intercalator, can be atargeting agent for double stranded DNA when the drug is covalentlylinked to an antibody and applied to DNA in a cell-free form.Doxorubicin, however, may not be considered a targeting agent withrespect to a cell while the drug is covalently linked to an antibodyunless the compound can be taken up by the cell. In the latter case,doxorubicin may have biological activity following uptake if the drugcan access DNA in the cell nucleus.

[0073] Biological agent functional components include, but are notlimited to, small molecule drugs (a pharmaceutical organic compound ofabout 5,000 daltons or less), organic molecules, proteins, peptides,peptidomimetics, glycoproteins, proteoglycans, lipids glycolipids,phospholipids, lipopolysaccharides, nucleic acids, proteoglycans,carbohydrates, and the like. Biological agents may be anti-neoplastic,anti-microbial, a hormone, an effector, and the like. Such compoundsinclude well known therapeutic compounds such as the anti-neoplasticagents paclitaxel, daunorubicin, doxorubicin, carminomycin,4′-epiadriamycin, 4-demethoxy-daunomycin, 11-deoxydaunorubicin,13-deoxydaunorubicin, adriamycin-14-benzoate, adriamycin-14-octanoate,adriamycin-14-naphthaleneacetate, vinblastine, vincristine, mitomycin C,N-methyl mitomycin C, bleomycin A₂, dideazatetrahydrofolic acid,aminopterin, methotrexate, cholchicine and cisplatin, and the like.Anti-microbial agents include aminoglycosides including gentamicin,antiviral compounds such as rifampicin, 3′-azido-3′-deoxythymidine (AZT)and acylovir, antifungal agents such as azoles including fluconazole,plyre macrolides such as amphotericin B, and candicidin, anti-parasiticcompounds such as antimonials, and the like. Hormones may include toxinssuch as diphtheria toxin, cytokines such as CSF, GSF, GMCSF, TNF,erythropoietin, immunomodulators or cytokines such as the interferons orinterleukins, a neuropeptide, reproductive hormone such as HGH, FSH, orLH, thyroid hormone, neurotransmitters such as acetylcholine, hormonereceptors such as the estrogen receptor. Also included are non-steroidalanti-inflammatories such as indomethacin, salicylic acid acetate,ibuprofen, sulindac, piroxicam, and naproxen, and anesthetics oranalgesics. Also included are radioisotopes such as those useful forimaging as well as for therapy.

[0074] Biological agent functional components for use in the targetingcompounds of the invention can be naturally occurring or synthetic.Biological agents can be biologically active in their native state, orbe biologically inactive or in a latent precursor state and acquirebiological or therapeutic activity when a portion of the biologicalagent is hydrolyzed, cleaved or is otherwise modified. The prodrug canbe delivered at the surface of a cell or intracellulary using antibodytargeting compounds of the invention where it can then be activated. Inthis regard, the biological agent can be a “prodrug,” meaning thatprodrug molecules capable of being converted to drugs (activetherapeutic compounds) by certain chemical or enzymatic modifications oftheir structure. In the prodrug approach, site-specific drug deliverycan be obtained from tissue-specific activation of a prodrug, which isthe result of metabolism by an enzyme that is either unique for thetissue or present at a higher concentration (compared with othertissues); thus, it activates the prodrug more efficiently.

[0075] Photodynamic treatment may be used to activate a prodrug bycleaving a photosenitive linker or by activating a photoresponsiveenzyme (acyl enzyme hydrolysis) as described previously (see U.S. Pat.Nos. 5,114,851 and 5,218,137). Photodynamic treatment also may be usedto rapidly inactivate a drug in sites where the drug activity is notdesired (e.g. in non-target tissues). Various means of covalentlymodifying a drug to form a prodrug are well known in the art.

[0076] Targeting agents may be covalently linked to the antibodycombining site directly or through the aid of a linker. An appropriatelinker can be chosen to provide sufficient distance between thetargeting agent and the antibody combining site in order for thetargeting agent to be able to bind to its target molecule. This distancedepends on several factors including, for example, the distance from theoutermost surface of the antibody combining site to the reactive sidechain in the combining site, and the nature of the targeting agent.Generally, the linker will be between about 5 to 10 angstroms (0.5 to 1nm) in length, with 10 or more angstroms (1.0 nm) being more preferred,although shorter linkers of about 3 angstroms (0.3 nm) in length may besufficient if the amino acid side chain is very near to the outermostportion of the combining site and/or the targeting agent or biologicalagent includes a segment that can function as a part of a linker.

[0077] Linker length may also be viewed in terms of the number of linearatoms (cyclic moieties such as aromatic rings and the like to be countedby taking the shortest route). Linker length under this measure isgenerally about 10 to 200 atoms and more typically about 30 or moreatoms, although shorter linkers of two or more atoms may be sufficientif the reactive amino acid side chain is very near to the outermostportion of the combining site. Generally, linkers with a linear stretchof at least about 9 atoms are sufficient. Other linker considerationsinclude effect on physical or pharmacokinetic properties of theresulting targeting compound or targeting agent-linker, solubility,lipophilicity, hydrophilicity, hydrophobicity, stability (more or lessstable as well as planned degradation), rigidity, flexibility,immunogenicity, modulation of antibody binding, chemical compatibilitywith targeting agent, ability to be incorporated into a micelle orliposome, and the like.

[0078] In targeting compounds where a linker is present between theantibody combining site, the targeting agent may be prepared by severalapproaches. In one approach, a targeting agent-linker compound and/orbiological agent-linker compound is synthesized with a linker thatincludes one or more reactive groups designed for covalent reaction witha side chain of an amino acid in the combining site of an antibody. Theagent-linker compound and antibody are combined under conditions wherethe linker reactive group forms a covalent bond with the amino acid sidechain.

[0079] In another approach, linking can be achieved by synthesizing anantibody-linker compound comprising an antibody and a linker wherein thelinker includes one or more reactive groups designed for covalentreaction with an appropriate chemical moiety of the targeting agent orbiological agent. The targeting agent or biological agent may need to bemodified to provide the appropriate moiety for reaction with the linkerreactive group. The antibody-linker and targeting agent and/orbiological agent are combined under conditions where the linker reactivegroup covalently links to the targeting and/or biological agent.

[0080] A further approach for forming an antibody targeting compound ofthe invention uses a dual linker design. In one embodiment, the anagent-linker compound is synthesized which comprises a targeting agentand/or a biological agent and a linker with a reactive group. Anantibody-linker compound is synthesized which comprises an antibody anda linker with a chemical group susceptible to reactivity with thereactive group of the agent-linker of the first step. These two linkercontaining compounds are then combined under conditions whereby thelinkers covalently link, forming the antibody targeting compound.

[0081] In another embodiment, an antibody-linker compound is synthesizedwhich comprises an antibody and a linker with a reactive group. Atargeting agent and/or biological agent-linker compound is preparedwhich comprises the agent and a linker with a chemical group susceptibleto reactivity with the reactive group of the antibody-linker of thefirst step. These two linker containing compounds are then combinedunder conditions whereby the linkers covalently link, forming theantibody targeting compound. “Susceptible” as used herein with referenceto a chemical moiety indicates that the chemical moiety will covalentlybond with a compatible reactive group. Thus, an electrophilic group issusceptible to covalent bonding with a nucleophillic group and viceversa.

[0082] As discussed, the linker may be first conjugated to the targetingagent and then the targeting agent-linker conjugated to the antibodycombining site. Alternatively, the linker may be conjugated first to theantibody combining site and the antibody-linker conjugated to thetargeting agent. Numerous means well known in the art can be used toattach a linker to the targeting agent or antibody combining site.Exemplary functional groups that can be involved in the linkage include,for example, esters, amides, ethers, phosphates, amino, keto, amidine,guanidine, imines, eneamines, phosphates, phosphonates, epoxides,aziridines, thioepoxides, masked or protected diketones (ketals forexample), lactams, haloketones, aldehydes, thiocarbamate, thioamide,thioester, sulfide, disulfide, phosphoramide, sulfonamide, urea,thioruea, carbamate, carbonate, hydroxamide, and the like.

[0083] The linker includes any atom from the group C, H, N, O, P, S, Si,halogen (F, Cl, Br, I) or a salt thereof. The linker also may include agroup such as an alkyl, alkenyl, alkynyl, oxoalkyl, oxoalkenyl,oxoalkynyl, aminoalkyl, aminoalkenyl, aminoalkynyl, sulfoalkyl,sulfoalkenyl, or sulfoalkynyl group, phosphoalkyl, phosphoalkenyl,phosphoalkynyl group. The linker also may include one or more ringstructures. As used herein a “ring structure” includes a carbocyclichomo or hetero mono or fused saturated or unsaturated ring structure.Combinations of the above groups and rings may also be present in thelinkers of the targeting compounds of the invention.

[0084] The general design of a embodiment of a unbranched linker for usein preparing targeting compounds of the present invention is shown inFIG. 2A. The linker is of the formula

X—Y—Z

[0085] Wherein X is a connecting chain, Y is a recognition group and Zis a reactive group. FIG. 2B-E shows various targeting agent-linkercompounds with the linker X, Y and Z portions identified. The linker maybe linear or branched. In some embodiments, the linker has a linearstretch of between 5-200 or 10-200 atoms although in other embodiments,longer linker lengths may be used. One or more targeting agents may belinked to X. In some embodiments, where more than one targeting agent islinked and a branched linker is used, some of the targeting agents maybe linked to different branches of the linker. However, it should beunderstood that linkersused in the compounds of the invention may haveone or more recognition groups, one or more reactive groups and one ormore connecting chains and combinations thereof. Connecting chains maybranch from another connecting chain or from a recognition group.

[0086] The connecting chain X of the linker includes any atom from thegroup C, H, N, O, P, S, Si, halogen (F, Cl, Br, I) or a salt thereof. Xalso may include a group such as an alkyl, alkenyl, alkynyl, oxoalkyl,oxoalkenyl, oxoalkynyl, aminoalkyl, aminoalkenyl, aminoalkynyl,sulfoalkyl, sulfoalkenyl, or sulfoalkynyl group, phosphoalkyl,phosphoalkenyl, phosphoalkynyl group. In some embodiments, X may includeone or more ring structures. In a preferred embodiment, X includes arepeating ether unit of between 2-100 units. Various embodiments of Xare shown in FIG. 9.

[0087] The recognition group Y of the linker is optional and if presentis located between the reactive group and the connecting chain. Inpreferred embodiments, Y is located from 1-20 atoms from Z. Although notwishing to be bound by any theory, it is believed that the recognitiongroup acts to properly position the reactive group into the antibodycombining site so that it may react with a reactive amino acid sidechain. FIG. 8 shows a variety of exemplary recognition groups with oneor more homo or hetero ring structures of five or six atoms. Larger ringstructures also may be used. One or more targeting agents may be linkedto Y. In some embodiments, a linker may be used to link the targetingagent to Y. In embodiments where two or more targeting agents are used,one or more can be attached to both X and Y. More than one targetingagent also can be attached to Y.

[0088] The linker reactive group Z includes any nucleophilic orelectrophilic group. In a preferred embodiment Z is capable of forming acovalent bond with a reactive side chain of an antibody. In someembodiments, Z includes one or more C═O, groups arranged to form adiketone, an acyl beta-lactam, an active ester, haloketone, a cyclohexyldiketone group, an aldehyde or maleimide. Other groups may includelactone, anhydride, and alpha-haloacetamide or an epoxide. Exemplarylinker electrophilic reactive groups that can covalently bond to areactive nucleophilic group (e.g. lysine or cysteine side chain) in thecombining site of an antibody include acyl beta-lactam, simple diketone,succinimide active ester, maleimide, haloacetamide with linker,haloketone, cyclohexyl diketone, aldehyde, amidine, guanidine, imine,eneamine, phosphate, phosphonate, epoxide, aziridine, thioepoxide, amasked or protected diketone (a ketal for example), lactam, sulfonate,and the like masked C═O groups such as imine, ketal, acetal and anyother known electrophilic group. A preferred linker reactive groupincludes one or more C═O, groups arranged to form a acyl beta-lactam,simple diketone, succinimide active ester, maleimide, haloacetamide withlinker, haloketone, cyclohexyl diketone, or aldehyde.

[0089] Z may be a group that forms a reversible or nonreversiblecovalent bond. In some embodiments, reversible covalent bonds may beformed using diketone Z groups such as those shown in FIG. 6. R₁ and R₂and R₃ in structures A-C of FIG. 6 represent substituents which can beC, H, N, O, P, S, Si, halogen (F, Cl, Br, I) or a salt thereof. Thesesubstituents also may include a group such as an alkyl, alkenyl,alkynyl, oxoalkyl, oxoalkenyl, oxoalkynyl, aminoalkyl, aminoalkenyl,aminoalkynyl, sulfoalkyl, sulfoalkenyl, or sulfoalkynyl group,phosphoalkyl, phosphoalkenyl,phosphoalkynyl group. R₂ and R₃ also couldfrom a ring structure as exemplified in structures B and C. X in FIG. 6could be a heteroatom. Other Z groups that form reversible covalentbonds include the diketone amidine, imine, and other reactive groupsshown in structures B and G of FIG. 7. FIG. 7 also includes thestructures of other preferred linker reactive groups.

[0090] Z reactive groups that form a nonreversible covalent bond withthe combining site of an antibody include structures D-G in FIG. 6 andstructures A, C and D of FIG. 7. Such structures are useful fornonreversibly attaching a targeting agent-linker to a reactivenucleophilic group (e.g. lysine or cysteine side chain) in the combiningsite of an antibody.

[0091] It should be understood that the above described reversible andnonreversible covalent linking chemistry can also be applied to link atargeting agent or biological agent to an antibody in the absence of alinker or to link a targeting agent or biological agent to a linker(e.g. to the connecting chain of the linker). For example, a targetingagent can be linked to a linker to form a targeting agent-linker byplacing a suitable reactive group Z type element such as an appropriatenucleophilic or electrophilic group on either the linker or thetargeting agent and a suitable reactive moiety such as an amino orsulfhydral group on the other of the two.

[0092] A preferred linker for use in targeting compounds of theinvention and for preparing targeting agent-linker compounds includes a1,3-diketone reactive group as Z. Another preferred linker is one wherethe connecting chain X includes a repeating ether unit of between 2-100units. Linkers in which the recognition group Y is present are preferredwith Y located preferably between 1-20 atoms from the reactive group Z.Such a linker attached to the core of an integrin targeting RGDpeptidomimetic moiety such as those described above, can have thestructure 28 as shown below where n is from 1-100 or more and preferablyis 1, 2, or 4, and more preferably is 3. In some embodiments, the linkeris a repeating polymer such as polyethylene glycol.

[0093] The linker reactive group or similar such reactive group that maybe inherent in the targeting agent, is chosen for use with a particularantibody. For example, a chemical moiety for modification by an aldolaseantibody may be a ketone, diketone, beta lactam, active esterhaloketone, lactone, anhydride, maleimide, alpha-haloacetamide,cyclohexyl diketone, epoxide, aldehyde, amidine, guanidine, imine,eneamine, phosphate, phosphonate, epoxide, aziridine, thioepoxide,masked or protected diketone (ketal for example), lactam, haloketone,aldehyde, and the like. A 1,3-diketone configuration such as thediketone shown in Compound SCS-873 (see below) or SCS-864 (see below),is especially preferred as a substrate for modification by an aldolaseantibody.

[0094] A linker reactive group chemical moiety (Z )suitable for covalentmodification by a reactive sulfhydryl group in an antibody may be adisulfide, aryl halide, maleimide, alpha-haloacetamide, isocyanate,epoxide, thioester, active ester, amidine, guanidine, imine, eneamine,phosphate, phosphonate, epoxide, aziridine, thioepoxide, masked orprotected diketone (ketal for example), lactam, haloketone, aldehyde,and the like. The chemical structures of various targeting agent-linkercompounds which include a linker with a 1,3 diketone as the reactivegroup are shown in FIGS. 2-5.

[0095] One of skill in the art will readily appreciate that reactiveamino acid side chains in antibodies may possess an electrophilic groupthat reacts with a nucleophilic group on the targeting agent or itslinker, whereas in other embodiments a reactive nucleophilic group in anamino acid side chain of a combining site of an antibody or an antibodyfragment reacts with an electrophilic group in a targeting agent orlinker. Thus, antibody or antibody fragment combining site side chainsmay be substituted with an electrophile (e.g., FIGS. 6 and 7) and thisgroup may be used to react with a nucleophile on the targeting agent orits linker (e.g., NH₂). In this embodiment, the antibody and targetingagent each have a partial linker with appropriate reactive moieties ateach end so that the two ends of the partial linker can form the fulllinker, thus creating the complete targeting compound.

[0096] One of skill in the art also will readily appreciate that two ormore targeting agents may be linked to a single antibody combining site.The two targeting agents may be the same or may be different withrespect to their specificity for a particular target. In one embodiment,each targeting agent may be linked to a separate reactive side chain ofan amino acid in the antibody combining site. In a preferred embodiment,the two targeting agents are attached to a branched or linear linkerwhich then links both targeting agents to the same reactive amino acidside chain in the antibody combining site. Each branch of a branchedlinker may in some embodiments comprise a linear stretch of between5-100 atoms. By way of example, the structures disclosed in FIGS. 3-5show embodiments of branched linkers with two targeting agents linked toa different branch of the linker, which has a 1,3-diketone as thereactive group. As shown in these embodiments, the branch point may bein the connecting chain or in the recognition group (if present).

[0097] “Antibody” as used herein includes immunoglobulins which are theproduct of B cells and variants thereof as well as the T cell receptor(TcR) which is the product of T cells and variants thereof. Animmunoglobulin is a protein comprising one or more polypeptidessubstantially encoded by the immunoglobulin kappa and lambda, alpha,gamma, delta, epsilon and mu constant region genes, as well as myriadimmunoglobulin variable region genes. Light chains are classified aseither kappa or lambda. Heavy chains are classified as gamma, mu, alpha,delta, or epsilon, which in turn define the immunoglobulin classes, IgG,IgM, IgA, IgD and IgE, respectively. Also subclasses of the heavy chainare known. For example, IgG heavy chains in humans can be any of IgG1,IgG2, IgG3 and IgG4 subclass.

[0098] A typical immunoglobulin structural unit is known to comprise atetramer. Each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kD) and one“heavy” chain (about 50-70 kD). The N-terminus of each chain defines avariable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. The terms variable lightchain(V_(L)) and variable heavy chain (V_(H)) refer to these light andheavy chains respectively.

[0099] Antibodies exist as full length intact antibodies or as a numberof welt characterized fragments produced by digestion with variouspeptidases or chemicals. Thus, for example, pepsin digests an antibodybelow the disulfide linkages in the hinge region to produce F(ab′)₂, adimer of Fab which itself is a light chain joined to V_(H)-CH₁ by adisulfide bond. The F(ab′)₂ may be reduced under mild conditions tobreak the disulfide linkage in the hinge region thereby converting theF(ab′)₂ dimer into an Fab′ monomer. The Fab′ monomer is essentially aFab fragment with part of the hinge region (see, Fundamental Immunology,W. E. Paul, ed., Raven Press, N.Y. (1993), for a more detaileddescription of other antibody fragments). While various antibodyfragments are defined in terms of the digestion of an intact antibody,one of skill will appreciate that any of a variety of antibody fragmentsmay be synthesized de novo either chemically or by utilizing recombinantDNA methodology. Thus, the term antibody, as used herein also includesantibody fragments either produced by the modification of wholeantibodies or synthesized de novo or antibodies and fragments obtainedby using recombinant DNA methodologies.

[0100] The T cell receptor (TcR) is a disulfide linked heterodimercomposed of α or β chains or, on a minority of T cells, γ or δ chains.The two chains are generally disulfide-bonded just outside the T cellplasma membrane in a short extended stretch of amino acids resemblingthe antibody hinge region. Each TcR chain is composed of oneAntibody-like variable domain (Vα or Vβ) and one constant domain (Cα orCβ). The full TcR has a molecular mass of about 95 kDa with theindividual chains varying in size from 35 to 47 kDa. Also encompassedwithin the meaning of TCR are portions of the receptor such as thevariable regions of this receptor that can be produced as a solubleprotein using methods well known in the art. For example, U.S. Pat. No.6,080,840 describes a soluble T cell receptor (TcR) prepared by splicingthe extracellular domains of a TcR to the glycosyl phosphatidylinositol(GPI) membrane anchor sequences of Thy-1. The molecule is expressed inthe absence of CD3 on the cell surface, and can be cleaved from themembrane by treatment with phosphatidylinositol specific phospholipase C(PI-PLC). The soluble TcR also may be prepared by coupling the TcRvariable domains to an antibody heavy chain CH₂ or CH₃ domain,essentially as described in U.S. Pat. No.5,216,132 or as soluble TcRsingle chains as described by Schusta et al. Nature Biotech. 18,754-759(2000) or Holler et al. Proc. Natl. Acad. Sci (USA) 97:5387-5392 (2000).The TcR “antibodies” as soluble products may be used in place ofantibody for making the compounds of the invention. The combining siteof the TcR can be identified by reference to CDR regions and otherframework residues using the same methods discussed above forantibodies.

[0101] Recombinant antibodies may be conventional full lengthantibodies, antibody fragments known from proteolytic digestion, uniqueantibody fragments such as Fv or single chain Fv (scFv), domain deletedantibodies, and the like. An Fv antibody is about 50 Kd in size andcomprises the variable regions of the light and heavy chain. A singlechain Fv (“scFv”) polypeptide is a covalently linked V_(H)::V_(L)heterodimer which may be expressed from a nucleic acid including V_(H)-and V_(L)-encoding sequences either joined directly or joined by apeptide-encoding linker. See Huston, et al. (1988) Proc. Nat. Acad. Sci.USA, 85:5879-5883. A number of structures for converting the naturallyaggregated, but chemically separated light and heavy polypeptide chainsfrom an antibody V region into an scFv molecule which will fold into athree dimensional structure substantially similar to the structure of anantigen-binding site. See, e.g. U.S. Pat. Nos. 5,091,513, 5,132,405 and4,956,778.

[0102] The combining site refers to the part of an antibody moleculethat participates in antigen binding. The antigen binding site is formedby amino acid residues of the N-terminal variable (“V”) regions of theheavy (“H”) and light (“L”) chains. The antibody variable regionscomprise three highly divergent stretches referred to as “hypervariableregions” or “complementarity determining regions” (CDRs) which areinterposed between more conserved flanking stretches known as “frameworkregions” (FRs). In an antibody molecule, the three hypervariable regionsof a light chain (LCDR1, LCDR2, and LCDR3) and the three hypervariableregions of a heavy chain (HCDR1, HCDR2 and HCDR3) are disposed relativeto each other in three dimensional space to form an antigen bindingsurface or pocket. The antibody combining site therefore represents theamino acids that make up the CDRs of an antibody and any frameworkresidues that make up the binding site pocket.

[0103] The identity of the amino acid residues in a particular antibodythat make up the combining site can be determined using methods wellknown in the art. For example, antibody CDRs may be identified as thehypervariable regions originally defined by Kabat et al. (see,“Sequences of Proteins of Immunological Interest,” E. Kabat et al., U.S.Department of Health and Human Services; Johnson, G and Wu, T T (2001)Kabat Database and its applications: future directions. Nucleic AcidsResearch, 29: 205-206; http://immuno.bme.nwa.edu). The positions of theCDRs may also be identified as the structural loop structures originallydescribed by Chothia and others, (see Chothia and Lesk, J. Mol. Biol.196, 901 (1987), Chothia et al., Nature 342, 877 (1989), and Tramontanoet al., J. Mol. Biol. 215, 175 (1990)). Other methods include the “AbMdefinition” which is a compromise between Kabat and Chothia and isderived using Oxford Molecular's AbM antibody modeling software (nowAccelrys) or the “contact definition” of CDRs by Macallum et al.,(“Antibody-antigen interactions: contact analysis and binding sitetopography,” J Mol Biol. Oct. 11, 1996;262(5):732-45). The followingchart identifies CDRs based upon various known definitions. Loop KabatAbM Chothia Contact LI L24-L34 L24-L34 L24-L34 L30-L36 L2 L50-L56L50-L56 L50-L56 L46-L55 L3 L89-L97 L89-L97 L89-L97 L89-L96 H1 H31-H35BH26-H35B H26-H32..34 H30-H35B (Kabat Numbering) H-1 H31-H35 H26-H35H26-H32 H30-H35 (Chothia Numbering) H2 H50-H65 H50-H58 H52-H56 H47-H58H3 H95-H102 H95-H102 H95-H10 H93-H101

[0104] General guidelines by which one may identify the CDRs in anantibody from sequence alone are as follows:

[0105] LCDR1:

[0106] Start—Approximately residue 24.

[0107] Residue before is always a Cys.

[0108] Residue after is always a Trp. Typically TRP is followed withTYR-GLN, but also may be followed by LEU-GLN, PHE-GLN, or TYR-LEU.

[0109] Length is 10 to 17 residues.

[0110] LCDR2:

[0111] Start—16 residues after the end of L1.

[0112] Sequence before is generally ILE-TYR, but also may be VAL-TYR,ILE-LYS, or ILE-PHE.

[0113] Length is generally 7 residues.

[0114] LCDR3:

[0115] Start—generally 33 residues after end of L2.

[0116] Residue before is a Cys.

[0117] Sequence after is PHE-GLY-X-GLY.

[0118] Length is 7 to 11 residues.

[0119] HCDR1:

[0120] Start—at approximately residue 26 (four residues after a CYS)[Chothia/AbM definition] Kabat definition starts 5 residues later.

[0121] Sequence before is CYS-X-X-X.

[0122] Residues after is a TRP, typically followed by VAL, but alsofollowed by ILE, or ALA.

[0123] Length is 10 to 12 residues under AbM definition while Chothiadefinition excludes the last 4 residues.

[0124] HCDR2:

[0125] Start—15 residues after the end of Kabat/AbM definition ofCDR-H1.

[0126] Sequence before typically LEU-GLU-TRP-ILE-GLY (SEQ ID NO. 1), buta number of variations are possible.

[0127] Sequence after is LYS/ARG-LEU/ILE/VAL/PHE/THR/ALA-THR/SER/ILE/ALA

[0128] Length is 16 to 19 residues under Kabat definition (AbMdefinition ends 7 residues earlier).

[0129] HCDR3:

[0130] Start—33 residues after end of CDR-H2 (two residues after a CYS).

[0131] Sequence before is CYS-X-X (typically CYS-ALA-ARG).

[0132] Sequence after is TRP-GLY-X-GLY.

[0133] Length is 3 to 25 residues.

[0134] The identity of the amino acid residues in a particular antibodythat are outside the CDRs, but nonetheless make up part of the combiningsite by having a side chain that is part of the lining of the combiningsite (i.e., it is available to linkage through the combining site), canbe determined using methods well known in the art such as molecularmodeling and X-ray crystallography. See e.g., Riechmann et al., (1988)Nature, 332:;323-327. The aldolase antibody mouse mAb 38C2, which has areactive lysine near to but outside HCDR3, is an example of such anantibody.

[0135] The reactive residue of the antibody combining site may benaturally associated with the antibody such as when the residue isencoded by nucleic acid present in the lymphoid cell first identified tomake the antibody. Alternatively, the amino acid residue may arise bypurposely mutating so as to encode the particular residue (see, e.g., WO01/22922 to Meares et al.). In another approach, the amino acid residueor its reactive elements (e.g., a nucleophilic amino group or sulfhydrylgroup) may be attached to an amino acid residue in the antibodycombining site. Thus, covalent linkage with the antibody occurring“through an amino acid residue in the combining site of the antibody” asused herein means that linkage can be directly to an amino acid residueof an antibody combining site or through a chemical moiety that islinked to a side chain of an amino acid residue of an antibody combiningsite

[0136] As discussed, antibodies that can be used in preparing theantibody targeting compounds of the invention require a reactive sidechain in the antibody combining site. A reactive side chain may bepresent or be placed by mutation in any antibody. Catalytic antibodiesare a preferred source of such antibodies. Such antibodies includealdolase antibodies, beta lactamase antibodies, esterase antibodies,amidase antibodies, and the like.

[0137] A reactive lysine in an antibody combining site may be covalentlylinked to a ketone, diketone, beta lactam, active ester haloketone,lactone, anhydride, maleimide, epoxide, aldehyde amidine, guanidine,imines, eneamines, phosphates, phosphonates, epoxides, aziridines,thioepoxides, masked or protected diketones (ketals for example),lactams, haloketones, aldehydes, and the like, associated with atargeting agent or linker-targeting agent. An exemplary and preferredsuch antibody is an aldolase antibody such as the mouse monoclonalantibody mAb 38C2 and other like catalytic antibodies as well assuitably humanized and chimeric versions of such antibodies. Mouse mAb38C2 is the prototype of a new class of catalytic antibodies that weregenerated by reactive immunization and mechanistically mimic naturalaldolase enzymes (Barbas et al., 1997, Science 278, 2085-2092). Througha reactive lysine, these antibodies catalyze aldol and retro-aldolreactions using the enamine mechanism of natural aldolases (Wagner etal., 1995, Science 270, 1797-1800; Barbas et al., 1997, Science 278,2085-2092; Zhong et al., 1999, Angew. Chem. Int. Ed. 38, 3738-3741;Karlstrom et al., 2000, Proc. Natl. Acad. Sci. U.S.A., 973878-3883). Inaddition to their versatility and efficacy in synthetic organicchemistry (e.g., Hoffmann et al., 1998, J. Am. Chem. Soc. 120, 2768-2779; Sinha et al., 1998, Proc. Natl. Acad. Sci. U.S.A. 95, 14603-14608),aldolase antibodies have been used to activate camptothecin,doxorubicin, and etoposide prodrugs in vitro and in vivo as ananti-cancer strategy (Shabat et al., 1999, Proc. Natl. Acad. Sci. U.S.A.96, 6925-6930 and ,2001, Proc. Natl. Acad. Sci. U.S.A. 98, 7528-7533).

[0138] In another example, the reactive amino acid of an antibodycombining site may be a reactive cysteine, serine or tyrosine residue.For cysteines, the resulting antibody may form a covalent linkage withmaleimide-containing components or other thiol-reactive groups such asiodoacetamides, aryl halides, disulfhydryls and the like. Reactivecysteines may be found in thioesterase catalytic antibodies as describedby Janda et al., Proc. Natl. Acad. Sci. (USA) 91:2532-2536, (1994). Forother esterase antibodies see Wirsching et al., Science 270:1775-82(1995). Reactive amino acid-containing antibodies may be prepared bymeans well known in the art including mutating an antibody combiningsite residue to encode for the reactive amino acid or chemicallyderivatizing an amino acid side chain in an antibody combining site witha linker that contains the reactive group.

[0139] Antibodies suitable for use herein may be obtained byconventional immunization, reactive immunization in vivo, or by reactiveselection in vitro, such as with phage display. Antibodies may beproduced in humans or in other animal species. Antibodies from onespecies of animal may be modified to reflect another species of animal.For example, human chimeric antibodies are those in which at least oneregion of the antibody is from a human immunoglobulin. A human chimericantibody is typically understood to have variable regions from anon-human animal, e.g. a rodent, with the constant regions from a human.In contrast, a humanized antibody uses CDRs from the non-human antibodywith most or all of the variable framework regions from and all theconstant regions from a human immunoglobulin. Chimeric and humanizedantibodies may be prepared by methods well known in the art includingCDR grafting approaches (see, e.g., U.S. Pat. Nos. 5,843,708; 6,180,370;5,693,762; 5,585,089; 5,530,101), chain shuffling strategies (see e.g.,U.S. Pat. No. 5,565,332; Rader et al., Proc. Natl. Acad. Sci. USA (1998)95:8910-8915), molecular modeling strategies (U.S. Pat. No. 5,639,641),and the like.

[0140] Unlike typical chemical derivatization of antibodies, thosederived from reactive immunization can be specifically labeled in theirbinding site at a defined position, facilitating the rapid andcontrolled preparation of a homogeneous product. In addition, unlikechemical derivatization of antibodies, those derived from reactiveimmunization with 1,3-diketones are reversible. Due to thisreversibility, a diketone derivative of a targeting compound bound tomAb 38C2 can be released from the antibody through competition with thecovalent binding hapten JW (Wagner et al., 1995, Science 270, 1797-800),or related compounds. This allows one to immediately neutralize theconjugate in vivo in case of an adverse reaction. Alternatively,non-reversible covalent linkage is possible such as with aldolaseantibodies and beta lactam derivatives of the targeting compound. Unliketypical anti-hapten antibodies, covalent diketone binding antibodieshave the advantage that the covalent linkage that is formed between thediketone and antibody is stable to large changes in pH, either extremesof low pH 3 or high pH 11. Such pH shifts do not release the targetingcompound from the antibody. This is an advantage for tumor targetingsince tumors typically exhibit reduced pH as compared to normal tissues.The added stability of covalent binding antibodies covalently linked totheir targeting agent should provide additional advantages in terms offormulation, delivery, and long term storage.

[0141] A targeting compound of the present invention can be made usingtechniques well known in the art. Typically, synthesis of a targetingagent which also is a functional component (biological agent) is thefirst step. The targeting agent (also functional component in this case)is then derivatized for linkage to a connecting component (the linker)which is then combined with the antibody. One of skill in the art willreadily appreciate that the specific synthetic steps used depend uponthe exact nature of the three components.

[0142] By way of example, as a first step, targeting agent-linkercompounds shown as Compounds 15 and 4, was made as shown in Schemes 1(FIG. 10) and 2 (FIG. 11), respectively, as derivatized versions of theintegrin targeting agents shown as Compounds 1 and 2, above. Compounds15 and 4 were derivatized (relative to Compounds 1 and 2) by addition ofa portion of the linker (connecting component). Scheme 3 (FIG. 12) showsadditional synthetic steps by which a complete linker with a diketonereactive moiety was added to derivatized targeting agent Compound 15 toobtain targeting compounds SCS-873 and SCS-1655.

[0143] Integrin targeting components shown as compounds 15 and 4 weresynthesized as shown in the FIG. 10 (Scheme 1) and FIG. 11 (Scheme 2),respectively. A linker with a diketone reactive moiety was added tothese targeting molecules as shown in Scheme 3 (FIG. 12) to formtargeting compound-linker molecules SCS-873 and SCS-1655. Synthesis ofSCS-873 was achieved starting from compound 14 in three steps. Compound14 was converted to 15 as shown in Scheme 1 and the crude product wasreacted with an N-hydroxysuccinimide (NHS)-ester of the diketonecompound 23 in CH3CN-DMF in the presence of Et3N. Purification oversilica gel (CH12Cl2-MeOH, 9:1) afforded pure SCS-873.

[0144] Compound SCS-1655 was synthesized from 14 in five steps (Schemes2 and 3). Deprotection of the BOC group in compound 14 followed byreaction with the NHS ester of the bivalent linker 24 afforded compound25, which was then deprotected and reacted with 23 as above to affordSCS-1655.

[0145] Synthesis of integrin targeting component-linker moleculesSCS-864 and SCS-789 is shown in Scheme 4 (FIG. 13). SCS-864 and SCS-789were each synthesized in one step from compound 4 (FIG. 13, scheme 4).Linking of Compound 4 was achieved with the appropriate activatedNHS-ester.

[0146] Targeting agent-linker compounds, such as SCS-864, SCS-873 andSCS-1655 where the linker includes a diketone reactive moiety, can beincubated with 0.5 equiv. of an aldolase antibody such as mAb 38C2 toproduce antibody targeting compounds. Additional examples are set forthbelow.

[0147] Also provided are targeting agent-linker compounds for covalentlylinking to a combining site of an antibody. The linker is of sufficientlength to allow the targeting agent to bind to the target molecule whenthe targeting agent is linked through the linker to an antibody. In someembodiments, the targeting agent-linker compound includes one or moretargeting agents specific for a target molecule with a linker of theformula X—Y—Z. The makeup of linker components X, Y and Z are asdescribed above. If two or more targeting agents are included in thetargeting agent-linker compound, the various targeting agents may beattached directly to the linker or the linker may be branched withtargeting agents attached to different linker branches.

[0148] Also provided is a targeting agent-linker compound that can benoncovalently associated with the combining site of an antibody. Thiscompound can be used in conjunction with a suitable antibody to form atargeting compound of the invention. Such targeting agent-linkercompounds comprise two or more targeting agents covalently linked via alinker to an antigen recognized by the antibody. The linker may linearor branched and should be of sufficient length to allow the targetingagent(s) to bind to the target molecule when the targeting agent(s) islinked through the linker to the antibody.

[0149] In some embodiments, the linker includes any of C, H, N, O, P, S,Si, F, Cl, Br, and I, or a salt thereof. The linker also may include agroup such as an alkyl, alkenyl, alkynyl, oxoalkyl, oxoalkenyl,oxoalkynyl, aminoalkyl, aminoalkenyl, aminoalkynyl, sulfoalkyl,sulfoalkenyl, or sulfoalkynyl group, phosphoalkyl, phosphoalkenyl,phosphoalkynyl group. The linker also may include one or more ringstructures. Combinations of the above groups and rings may also bepresent in the linkers of the targeting compounds of the invention. Insome embodiments, the linker has a linear stretch of between 2-200 atomsalthough in other embodiments, longer linker lengths may be used. One ormore targeting agents may be linked to the linker and if a branchedlinker is used, some of the targeting agents may be linked to differentbranches of the linker.

[0150] In some embodiments, the targeting agent of the targetingagent-linker compound is biologically active while in other embodiments,the targeting agent-linker compound further includes a separatebiological agent, which is preferably covalently linked to the targetingagent. In some embodiments, the biological agent may be linked to thetargeting agent or to the linker using essentially the same approachesused to link the targeting agent to the linker or using other approacheswell known in the art.

[0151] The antigen of the linker can be any antigen which can be boundby an available antibody. Antigens are well known in the art andinclude, an organic compound, a drug, a biomolecule such as a protein,peptide, peptidomimetic, glycoprotein, proteoglycan, lipid, glycolipid,nucleic acid, carbohydrates, and the like as well as combinations ofthese molecules.

[0152] The present invention also includes methods of modifying thecombining site of an antibody to generate binding specificity for aparticular target molecule. Such methods include covalently linking areactive amino acid side chain in the combining site of the antibody toa chemical moiety on a linker of a targeting agent-linker compound wherethe targeting agent is specific for the target molecule. The chemicalmoiety of the linker is sufficiently distanced from the targeting agentso that the targeting agent can bind to the target molecule when thetargeting agent-linker compound is covalently linked to the antibodycombining site. Typically, the antibody will not be considered specificfor the target molecule. In a preferred embodiment, the antibody priorto covalent linking would have an affinity for the target molecule ofless than about 1×10⁻⁵ moles/liter. However, after the antibody iscovalently linked to the targeting agent-linker compound, the modifiedantibody preferably has an affinity for the target molecule of at leastabout 1×10⁻⁶ moles/liter, more preferably at least about 1×10⁻⁷moles/liter, even more preferably at least 1×10⁻⁸ moles/liter, yet evenmore preferably at least 1×10⁻⁹ moles/liter, most preferably at leastabout 1×10⁻¹⁰ moles/liter.

[0153] The present invention also includes methods of altering at leastone physical or biological characteristic of a targeting agent,biological agent or linker. The methods include covalently linking thetargeting agent or biological agent to the combining site of an antibodyas described above. In some embodiments, the targeting agent orbiological agent is linked to the antibody combining site though alinker, the characteristics of which are described above. The method isparticularly useful for linking small targeting or biological agents of5 Kd or less. However, the method also works for larger such molecules.Characteristics of the targeting agent or biological agent can includebinding affinity, susceptibility to degradation, such as by proteases,pharmocokinetics, pharmacodynamics, immunogenicity, solubility,solubility, lipophilicity, hydrophilicity, hydrophobicity, stability(more or less stable as well as planned degradation), rigidity,flexibility, modulation of antibody binding, and the like.

[0154] As used herein, pharmacokinetics refers to the concentration anadministered compound in the serum over time. Pharmacodynamics refers tothe concentration of an administered compound in target and nontargettissues over time and the effects on the target tissue (efficacy) andthe non-target tissue (toxicity). Improvements in, for example,pharmacokinetics or pharmacodynamics can be designed for a particulartargeting agent or biological agent such as by using labile linkages orby modifying the chemical nature of any linker (changing solubility,charge, etc.).

[0155] The biological characteristic of an antibody targeting compoundof the invention may be modified to obtain improved pharmaceutical orother characteristics. This may be achieved by altering one or morechemical characteristics of the targeting agent or biological agent, thelinker or the antibody. A preferred approach is to chemically modify oneor more chemical characteristics of the linker. By altering chemicalcharacteristics of the compound including the linker, one can obtainimproved features such as improvement in pharmockinetics,pharmacodynamics, solubility, immunogenicity and the like.

[0156] The targeting compounds of the present invention have many uses.For example, the antibody portion of a targeting compound may generallyextend the half-life of a smaller sized targeting agent in vivo. Also,the biological potency of a particular targeting agent may be increasedby the addition of effector function(s) provided by the antibody portionof the targeting compound (e.g., complement mediated effectorfunctions). In addition, the targeting agent, through its increased sizeconferred by linkage to the antibody, may enable the targeting agent tofunction as a competitive inhibitor in situations where it wouldotherwise fail to do so. Thus, in one aspect, the invention provides amethod for increasing the effective circulating half-life of a targetingagent. The method includes linking the targeting agent to an antibodyusing a linking group as set forth above. In another aspect, theinvention provides a method of redirecting an antibody to a specifictarget. The method includes linking an antibody to a targeting agentthrough a linker as set forth above.

[0157] The invention also provides a method of treating or preventing adisease or condition in an individual wherein said disease or conditioninvolves cells, tissue or fluid that expresses a target molecule. Themethod includes administering to a subject such as a patient, atherapeutically effective amount of a targeting compound of theinvention. The subject may be an animal such as a mammal. In someembodiments, the subject is a human. The compound may include abiological agent that is the same or is distinct from the targetingagent and which may take any of the forms or activities describedherein. In some preferred embodiments, the target molecule is anintegrin and the disease is a carcinoma. The association of integrinexpression in carcinomas is well known in the art (See, e. U.S. Pat.Nos. 5,753,230 and 5,766,591, the disclosures of which are incorporatedherein by reference). For therapeutic use in humans, a human, humanized,or human chimeric antibody is a preferred as the antibody component ofthe targeting compound. An antibody with a human IgG4 constant regionalso is preferred if agonist activity is desired.

[0158] In addition to therapeutic applications, antibody targetingcompounds of the invention may also be used for the imaging of cellssuch as tumor cells or tissues (e.g., an extracellular matrixbiomolecule) as is well known in the art. Accordingly, provided is amethod of imaging cells or tissue (e.g., an extracellular matrixbiomolecule) in an individual. In such methods, the cells or tissueexpresses a target molecule. The method includes administering to asubject an antibody targeting compound of the invention linked to adetectable label. A detectable label for use in such methods can be aradioisotope or may be a non-radioisotope such as may be used in nuclearmagnetic resonance (NMR) imaging. In the latter case, one may link theantibody targeting agent to chelates e.g.,diethylenetriaminepentaacetate (DTPA) of the paramagnetic metalgadolinium essentially as described in Simkins et al., Nat. Med.,4(5):623-6 (1998).

[0159] The binding of a mixture of SCS-873 and 38C2 to human Karposi'ssarcoma SLK cells was studied. SCS-873 effectively mediated cell surfacebinding of 38C2. No binding of 38C2 was detectable in the absence ofSCS-873. Control experiments confirmed that the 1,3-diketone moiety isrequired for binding of SCS-873 to 38C2. After independent i.p. and i.v.injections, respectively, SCS-873 and 38C2 form an integrin α_(v)β₃targeting conjugate in vivo. In these experiments, the circulatoryhalf-life of SCS-873 was extended by more than two orders of magnitudethrough binding to 38C2. Combination of SCS-873 and 38C2 effectivelyinhibited tumor growth in a mouse model of human Karposi's sarcoma,whereas either SCS-873 or 38C2 alone were less effective or noteffective at all.

[0160] The present invention also provides methods of targeting abiological activity to cells, tissue (e.g., an extracellular matrixbiomolecule) or a biolomolecule in the fluid of a subject. The methodincludes administering to the subject, a targeting compound thatincludes a targeting agent specific for the cells, tissue extracellularmatrix biomolecule or fluid biomolecule. The targeting agent iscovalently linked to an amino acid residue in the combining site of anantibody. In some embodiments, a linker is used to link the targetingagent to the antibody. The targeting agent is not an antibody. In someembodiments, the compound has a biological activity while in otherembodiments, an biologically active molecule that is not the targetingagent is included as a component of the compound. Alternatively, thecomponent parts of the targeting compound may be separately administeredand then form the covalent compound in vivo. In such a method, thetargeting agent may include a linker/reactive moiety or the antibodycombining site may be suitably modified to covalently link to thetargeting agent.

[0161] A targeting compound of the present invention can be administeredas a pharmaceutical or medicament that includes a targeting compound ofthe invention formulated with a pharmaceutically acceptable carrier.Accordingly, the compounds may be used in the manufacture of amedicament or pharmaceutical composition. Pharmaceutical compositions ofthe invention may be formulated as solutions or lyophilized powders forparenteral administration. Powders may be reconstituted by addition of asuitable diluent or other pharmaceutically acceptable carrier prior touse. Liquid formulations may be buffered, isotonic, aqueous solutions.Powders also may be sprayed in dry form. Examples of suitable diluentsare normal isotonic saline solution, standard 5% dextrose in water, orbuffered sodium or ammonium acetate solution. Such formulations areespecially suitable for parenteral administration, but may also be usedfor oral administration or contained in a metered dose inhaler ornebulizer for insufflation. It may be desirable to add excipients suchas polyvinylpyrrolidone, gelatin, hydroxy cellulose, acacia,polyethylene glycol, mannitol, sodium chloride, sodium citrate, and thelike.

[0162] Alternately, compounds may be encapsulated, tableted or preparedin an emulsion or syrup for oral administration. Pharmaceuticallyacceptable solid or liquid carriers may be added to enhance or stabilizethe composition, or to facilitate preparation of the composition. Solidcarriers include starch, lactose, calcium sulfate dihydrate, terra alba,magnesium stearate or stearic acid, talc, pectin, acacia, agar orgelatin. Liquid carriers include syrup, peanut oil, olive oil, salineand water. The carrier may also include a sustained release materialsuch as glyceryl monostearate or glyceryl distearate, alone or with awax. The amount of solid carrier varies but, preferably, will be betweenabout 20 mg to about 1 g per dosage unit. The pharmaceuticalpreparations are made following the conventional techniques of pharmacyinvolving milling, mixing, granulating, and compressing, when necessary,for tablet forms; or milling, mixing and filling for hard gelatincapsule forms. When a liquid carrier is used, the preparation may be inthe form of a syrup, elixir, emulsion, or an aqueous or non-aqueoussuspension. For rectal administration, the invention compounds may becombined with excipients such as cocoa butter, glycerin, gelatin orpolyethylene glycols and molded into a suppository.

[0163] Compounds of the invention may be formulated to include othermedically useful drugs or biological agents. The compounds also may beadministered in conjunction with the administration of other drugs orbiological agents useful for the disease or condition that the inventioncompounds are directed.

[0164] As employed herein, the phrase “an effective amount,” refers to adose sufficient to provide concentrations high enough to impart abeneficial effect on the recipient thereof. The specific therapeuticallyeffective dose level for any particular subject will depend upon avariety of factors including the disorder being treated, the severity ofthe disorder, the activity of the specific compound, the route ofadministration, the rate of clearance of the compound, the duration oftreatment, the drugs used in combination or coincident with thecompound, the age, body weight, sex, diet, and general health of thesubject, and like factors well known in the medical arts and sciences.Various general considerations taken into account in determining the“therapeutically effective amount” are known to those of skill in theart and are described, e.g., in Gilman et al., eds., Goodman AndGilman's: The Pharmacological Bases of Therapeutics, 8th ed., PergamonPress, 1990; and Remington's Pharmaceutical Sciences, 17th ed., MackPublishing Co., Easton, Pa., 1990. Dosage levels typically fall in therange of about 0.001 up to 100 mg/kg/day; with levels in the range ofabout 0.05 up to 10 mg/kg/day are generally applicable. A compound canbe administered parenterally, such as intravascularly, intravenously,intraarterially, intramuscularly, subcutaneously, or the like.Administration can also be orally, nasally, rectally, transdermally orinhalationally via an aerosol. The composition may be administered as abolus, or slowly infused.

[0165] The administration of an antibody-targeting agent conjugate to animmunocompetent individual may result in the production of antibodiesagainst the conjugate. Such antibodies may be directed to the antibodyitself, such as the variable region including the antibody idiotype aswell as to the targeting agent or any linker used to conjugate thetargeting agent to the antibody. Reducing the immunogenicity of theantibody-targeting agent conjugate can be addressed by methods wellknown in the art such as by attaching long chain polyethylene glycol(PEG)-based spacers, and the like, to the antibody-targeting agent. Longchain PEG and other polymers are known for their ability to mask foreignepitopes, resulting in the reduced immunogenicity of therapeuticproteins that display foreign epitopes (Katre et al., 1990, J. Immunol.144, 209-213; Francis et al., 1998, Int. J. Hematol. 68, 1-18). Asnoted, PEG can be a linker as well, thus providing both linker functionand reduced immunogenicity in a targeting compound of the invention.Alternatively, or in addition, the individual administered theantibody-targeting agent conjugate may be administered animmunosuppressent such as cyclosporin A, anti-CD3 antibody, and thelike.

[0166] A method of screening a chemical library for agonists orantagonists of a receptor is further provided. The method includeslinking individual members of the chemical library to the combining siteof an antibody and then testing the antibody linked library for bindingto the receptor or for inhibition of binding between the receptor and aligand for the receptor. By this approach, the present antibodytargeting compounds provide a new format for high throughput screeningto identify candidate small molecule chemicals such as drugs peptidespeptidomimetics, organic compounds, and the like, that function forexample, as antagonists or agonists. The relative small size of a usefulcandidate chemical molecule typically requires indirect screening suchas in displacement or competition formats. As provided herein, one canbuild the chemical library on an antibody format, by linking individualdrugs in the library to a combining site of an antibody.

[0167] Antibody combining site-tagged libraries may be prepared bysynthesizing chemical candidates with a suitable linker comprising aparticular linker moiety designed for covalent interaction with aparticular antibody. Such linkers may include a diketone moiety to beused in conjunction with an aldolase antibody that includes a reactivelysine in the combining site. One skilled in the art would readilyunderstand that other linkers and linker moieties (e.g., biotin) whichhave been described herein are clearly useful for this purpose.

[0168] Antibody combining site-tagged chemical libraries thus preparedcan be used, for example, in receptor assays or cell bioassays wherebinding of each compound in the library may be monitored by detectingthe linked antibody. Detection of the antibody portion of each compoundmay be accomplished by methods of antibody detection well known in theart. For example, the antibody may be linked to a detectable moiety suchas an enzyme, fluorophore, radioisotope, and the like. Indirect systemscan also be used such as biotin-streptavidin. Libraries can be screenedon cells or impure antigens such as viral lysates as well as on purifiedantigens. For example, libraries can be tested for binding or inhibitionof binding using as the target, lysates run on protein gels, with theanalysis focussed on a particular gel band. In the case where thereceptor is expressed on a cell, binding or inhibition of binding can bedetermined by detecting cellular signaling events occurring (or notoccurring as in the case of inhibition) downstream of said binding orinhibition of binding. Downstream cellular signaling can be detectedwith the aid of a reporter gene as is well known in the art (see, e.g.,U.S. Pat. Nos. 5,618,720 and 5,670,113).

[0169] Screening of antibody tagged chemical libraries can be readilyadapted for use with high throughput instruments. Screening may be donein vitro or in vivo. Furthermore, a biological display library such as apeptide phage library may be used to prepare an antibody combiningsite-tagged library. In such cases, the site of attachment of the linkermoiety (e.g., diketone) can be the fusion point of the library to thebiological carrier.

[0170] Also provided is an immunoassay method for determining the amountof analyte in a sample. Such methods include:

[0171] (a) forming, in a medium containing a sample, a complex betweenthe analyte and at least one antibody specific for the analyte;

[0172] (b) analyzing the medium to detect the amount of the complex; and

[0173] (c) relating the amount of the complex to the amount of analytein the sample.

[0174] Such methods may also include forming the complex with at leastone antibody that is specific for the analyte. The specificity of theantibody is provided by a non-antibody targeting agent specific for theanalyte which is covalently linked to a reactive amino acid in thecombining site of the antibody. Thus, the antibody targeting compoundsof the invention can be used in immunoassays for detecting and measuringthe amount of an analyte in a sample as has been done previously withconventionally prepared polyclonal or monoclonal antibodies. Such assaysare well known in the art and include RIA, EIA, Western, ELISA, and thelike. The assay formats may be competitive or non-competitive and may bedirect or indirect. The antibody targeting compound can be used in theliquid phase and/or can be bound to a solid phase carrier. Carriersinclude glass, polystyrene, polypropylene, polyethylene, dextran, nylon,natural and modified cellulose, polyacrylamide, agarose, magnetite, andthe like. The nature of the carrier can be either soluble or insoluble.The antibody targeting compound may be detectably labeled in any ofvarious ways well known in the art. U.S. Pat. Nos. 4,659,678; 4,780,423;and 4,298,685 are exemplary of such assays.

[0175] Viewed in general terms, the amount of an analyte in a sample canbe determined by forming, in a medium containing the sample, a complexbetween the analyte and at least one antibody specific for the analyte.The medium is then analyzed to determine the amount of the complex thatis formed. Finally, the amount of complex formed is then related to theamount of analyte in the sample. As already described, this generalapproach can take many forms such as direct and indirect, homogenous orheterogeneous, and competitive and noncompetitive. In all cases, theantibody targeting compounds of the invention may be used to replacefunctions provided by conventionally prepared antibodies.

[0176] Also provided is a direct or indirect binding assay where thepresence of an analyte is determined using an antibody specific for theanalyte. In such methods, the presence of the analyte is determinedusing an antibody specific for the analyte. The antibody specificityresults from a non-antibody targeting agent that is specific for theanalyte, and the targeting agent is covalently linked to a reactiveamino acid in the combining site of the antibody. Thus,antibody-targeting compounds of the invention can be used in qualitativeassays in place of conventionally prepared antibodies.

[0177] It would be readily evident that the compounds of the inventionfind use not only in human medical therapy and diagnosis but also inveterinary, agricultural, environmental and other disciplines.

[0178] Also provided are methods of inhibiting or reducing the abilityof a targeting agent or biological agent to cross a cell membrane. Inthese methods an antibody targeting compound is formed by covalentlylinking the combining site of an antibody that does not itself cross thecell membrane to the targeting agent or biological agent, whereinlinkage of said antibody to said targeting agent or biological agentreduces or inhibits the ability of the agent to cross the cell membrane.Antibodies that are not directed to cell surface internalizing receptorsare a preferred source of antibodies that do not cross cell membranes.

[0179] Further provided are methods of mediating intracellular deliveryof a intracellularly active drug. In these methods, an antibodytargeting compound is prepared wherein said compound includes one ormore targeting agents or one or more biological agents or bothcovalently linked via a linker to the combining site of the antibody.The targeting agents or biological agents are characterized in that theybind to a cell receptor and mediate internalization of the agent. Theantibody targeting compound also includes a drug that is activeintracellularly. Intracellular drug delivery occurs when a cellexpressing the receptor contacts the antibody targeting compound. Thecontacting results in internalization of the antibody targeting agentand delivery of said drug intracellularly.

[0180] This approach uses takes advantage of receptor mediatedendocytosis (i.e., receptor mediated internalization) to deliver theantibody targeting compound intracellularly. Cell surface receptors thatmediate internalization of binding ligands are well known in the art andinclude, for example, integrins, HER2, EGF receptor, folic acidreceptor, and the like. Internalization assays are readily available andcan be evaluated using fluorescent detection methods.

[0181] In some embodiments, the intracellularly active drug is a prodrugthat becomes active when said drug contacts an intracellularcompartment. The antibody targeting compound may include anintracellular trafficking signal to direct the internalized antibodytargeting compound to a particular intracellular compartment. Manyproteins contain one or more targeting sequences that serve as atrafficking signal or address to target the protein to the correctintracellular site. Receptors at the destination also may be involved inthe trafficking process.

[0182] The sequences that direct proteins and other compounds todifferent intracellular sites such as endoplasmic reticulum, endosome,golgi, or nucleus, and the like, are well known in the art. For example,endoplasmic reticulum trafficking signals include a KDEL or KKXXsequence, golgi trafficking signals include a GRIP domain (see Munro etal., Curr Biol 9: 377-379, 1999), lysosomal trafficking signals (fromgolgi) include mannose-6-phosphate modified oligosaccharides, andnuclear localization trafficking signals which include one or two shortpositively charged sequences, e.g., lysine or arginine rich (see, Pencoet al. Biotech Appl Biochem 34:151-159 2001).

[0183] The versatility of the invention is illustrated by the followingExamples which illustrate preferred embodiments of the invention and arenot limiting of the claims or specification in any way.

EXAMPLE 1 Antibody Targeting Compound Comprising an RGD PeptidomimeticTargeting Agent Covalently Linked to the Combining Site of AldolaseMonoclonal Antibody 38C2

[0184] An integrin targeting compound was formed based on the formationof a reversible covalent bond between a diketone linker derivative of anRGD peptidomimetic and the reactive lysine of mouse mAb 38C2. Mouse mAb38C2 is the prototype for a new class of catalytic antibodies generatedby reactive immunization and mechanistically mimic natural aldolaseenzymes (Barbas et al., Science 278, 2085-2092, 1997). Through areactive lysine, these antibodies catalyze aldol and retro-aldolreactions using the enamine mechanism of natural aldolases (Wagner etal., Science 270, 1797-1800, 1995; Barbas et al., Science 278,2085-2092, 1997; Zhong et al., Angew. Chem. Int. Ed. 38, 3738-3741,1999). In addition to their versatility and efficacy in syntheticorganic chemistry, aldolase antibodies have been used in the activationof camptothecin, doxorubicin, and etoposide prodrugs in vitro and invivo as an anti-cancer strategy (Shabat et al., Proc. Natl. Acad. Sci.U.S.A. 96, 6925-6930, 1999); Shabat, D. et al. Proc. Natl. Acad. Sci.U.S.A. 98, 7528-7533, 2001). Yet another feature of these antibodies,namely their ability to bind diketones covalently, has remained largelyunexplored.

[0185] The RGD peptidomimetic used (see Compound 1) is specific forhuman integrin with a high binding affinity for α_(v)β₃ at 0.9 nM andα_(v)β₅ at 0.6 nM (specificity exhibited by minimal a_(IIb)b₃ binding)(Miller et al., supra). A diketone linker modified version of Compound1, designated SCS-873, was prepared as described above.

[0186] A peptidomimetic RGD antagonist with known activity for bothα_(v)β₃ or α_(v)β₅ binding is desirable because some of these compoundsbind both murine and human integrins. Such species cross reactivityaffords preclinical in vivo studies in animal angiogenesis models priorto human trials. In addition, the targeting compound may be used for thetherapy of Kaposi's sarcoma which is associated with α_(v)β₃ integrin.

[0187] SCS-873 was linked to antibody 38C2 by the following procedure:One milliliter antibody 38C2 in phosphate buffered saline (10 mg/ml) wasadded to 12 microliters of a 10 mg/mL stock solution of SCS-873 and theresulting mixture was maintained at room temperature for 2 hours priorto use.

[0188] The binding of a mixture of SCS-873 and 38C2 to SLK cells wasevaluated. SCS-873 effectively mediated cell surface binding of 38C2. Nobinding of 38C2 was detectable in the absence of SCS-873. Controlexperiments confirmed that the diketone moiety of the linker is requiredfor binding of SCS-873 to 38C2. It was determined that SCS-873 retainsthe integrin specificity of the integrin targeting component, i.e., nobinding to a_(IIb)b₃ in ELISA was detected while binding to α_(v)β₃ andα_(v)β₃ was found to be strong. Independent i.p. and i.v. injections ofthe targeting compound prepared with SCS-873 and 38C2 versus eachcomponent alone into mice demonstrated integrin targeting in vivo. Inthese experiments, the serum half-life of SCS-873 was extended by morethan two orders of magnitude through binding to 38C2. Free SCS-873 notbound to antibody had a serum half-life of only minutes while thecombination of antibody and small molecule could be detected in theserum sampled from eye bleeds after several days.

EXAMPLE 2 Antibody Targeting Compound Comprising IL-4 as Targeting AgentCovalently Linked to the Combining Site of Aldolase Monoclonal Antibody38C2

[0189] Kaposi's sarcoma tumor cells, among other human epithelial tumorcells, express interleukin-4 (IL-4) receptors that can be targeted witha recombinant chimeric protein consisting of IL-4 and a truncated formof bacterial toxin called Pseudomonas exotoxin (Husain et al., 1999,Nat. Med. 5, 817-822). Based on these studies, an IL-4 targetingcompound for targeting mAb 38C2 to Kaposi's sarcoma tumor cells isprepared. A linker with a diketone reactive group is conjugated to alysine side chain of IL-2 using a lysine reactive moiety such asN-hydroxysuccinimide (NHS). Alternatively, a recombinant IL-4 with anadded free cysteine is used for conjugation to cysteine reactivemoieties such as maleimide. To reduce immunogenicity associated with thelinker portion of the targeting compound, the spacer (i.e. linkerconnecting chain) between the diketone reactive group on one end and theNHS or maleimide group on the other, is a polyethylene glycol (PEG)chain. Long chain PEG and other polymers are known for their ability tomask foreign epitopes, resulting in the reduced immunogenicity oftherapeutic proteins that display foreign epitopes (Katre et al., 1990,J. Immunol. 144, 209-213; Francis et al., 1998, Int. J. Hematol. 68,1-18). Not more than one to two diketones should be conjugated to theIL-4 in order to avoid clearance of cross-linked antibodies (Rehlaenderand Cho, 1998, Pharm. Res. 15, 1652-1656). Other interleukins such asIL-2 can be used in place of IL-4 as the targeting agent. While IL-4 canbe used primarily as a targeting module, an enhancement of itspharmacological effect (Lussow et al., 1996, Transplantation 62,1703-1708) may result from IL-2 receptor triggering due to the prolongedserum half-life of the interleukin obtained through its linkage to anantibody.

EXAMPLE 3 Antibody Targeting Compound Comprising VEGF-R2 Binding Peptideas Targeting Agent Covalently Linked to the Combining Site of AldolaseMonoclonal Antibody 38C2

[0190] Vascular endothelial growth factor (VEGF) is a key modulator oftumor angiogenesis. Induced by hypoxia, VEGF expression is upregulatedthrough the induction of VEGF mRNA transcription in the tumor. Followingproduction and release by the tumor, VEGF diffuses to endothelial cellsof nearby preexisting blood vessels, which display VEGF receptors(VEGFR). VEGF binds to two tyrosine kinase receptors, VEGFR-1 andVEGFR-2, which are expressed predominantly on endothelial cells.Activation of endothelial cells is associated with the binding of VEGFto VEGFR-2, whereas VEGFR-1 probably functions as a decoy receptor thatregulates the local concentration of VEGF (Neufeld et al., 1999, FASEBJ. 13, 9-22). Following activation, the endothelial cells proliferate,migrate directionally toward the tumor, and eventually roll up andinterconnect to form new blood vessels. Anti-angiogenic drugs thatinterfere with the interaction of VEGF and VEGR-2 are promisingcandidates for cancer therapy (Klohs and Hamby, 1999, Curr. Opin.Biotechnol. 10, 544-549). Binétruy-Tournaire et al. (2000, EMBO J. 19,1525-1533) identified the VEGFR-2 binding linear peptide ATWLPPR (SEQ IDNO: 2) through phage display of peptide libraries. ATWLPPR (SEQ ID NO:2) effectively interfered with VEGF binding to VEGFR-2 and inhibitedVEGF-mediated angiogenesis.

[0191] An antibody targeting compound comprising VEGF-R2 binding peptideis prepared by synthesizing the peptide with an additional Cys residueat the amino or carboxy terminus, resulting in a peptide with thesequence ATWLPPRC (SEQ ID NO: 3) and CATWLPPR (SEQ ID NO: 4),respectively. These thiol-modified peptides are reacted with amaleimide/diketone linker (FIG. 14) to produce peptide-linker-diketo anddiketo-linker-peptide. Incubation of these diketone derivatives withmAb38C2 results in a covalent linkage between the VEGFR-2 peptide andthe antibody combining site. The resulting antibody—VEGFR-2 targetingcompound is used to target endothelial cells that express VEGFR-2 suchas in tumor angiogenesis. The compound prolongs the half-life of thepeptide and equips it with antibody effector function.

EXAMPLE 4 Antibody Targeting Compound Comprising Neutralizing RNAAptamer as Targeting Agent Covalently Linked to the Combining Site ofAldolase Monoclonal Antibody 38C2

[0192] Using the process of SELEX (Systematic Evolution of Ligands byExponential Enrichment), RNA and DNA aptamers to a variety of moleculartargets have been generated (Jayasena, 1999, Clin. Chem. 45, 1628-1650).For example, 2′ fluoropyrimidine RNA aptamers that include about 25nucleotides and that bind VEGF with an affinity in the 100-pM range weredescribed (Ruckman et al., 1999, J Biol. Chem. 32, 20556-20567). Likethe peptide described in the previous example, the aptamers were foundto interfere with the interaction of VEGF and VEGFR-2.

[0193] An antibody targeting compound comprising VEGF RNA aptamer isprepared using commercially available thiol-derivatized nucleotides suchas 5′-phosphorothioate. A phosphorothioate group is a modified phosphategroup with one of the oxygen atoms replaced by a sulfur atom. Thethiol-modified nucleotide within the RNA aptamer is reacted with amaleimide diketone (e.g., FIG. 14) to produce an RNA aptamertargeting-diketone linker compound. Alternatively, a primary amino groupis introduced into the RNA aptamer using commercially available aminomodifiers. A nucleotide labeled with a primary amino group within theRNA aptamer is reacted with a linker that has N-hydroxysuccinimidediketone as the reactive group. Incubation of the diketone derivativeswith mAb38C2 results in a covalent linkage between the RNA aptamer andthe antibody combining site. The resulting antibody—RNA aptamer VEGFR-2targeting compound is used to target endothelial cells that expressVEGFR-2 such as in tumor angiogenesis. The compound prolongs thehalf-life of the RNA aptamer and equips it with antibody effectorfunction.

EXAMPLE 5 Antibody Targeting Compound Comprising Folate as TargetingAgent Covalently Linked to the Combining Site of Aldolase MonoclonalAntibody 38C2

[0194] The folate receptor mediates the uptake of folic acid into cellsby endocytosis. It is overexpressed on a variety of epithelial tumorcells (Leamon and Low, 2001, Drug Discov. Today 6, 44-51). For example,greater than 90% of ovarian carcinomas express the folate receptor(Sudimack and Lee, 2000, Adv. Drug Deliv. Rev. 41, 147-162). Mabsdirected to the folate receptor, for example Mov 18 and Mov19, have beenevaluated as drugs for ovarian cancer therapy (Coney et al., 1994,Cancer Res. 54, 2448-2455; Molthoff et al., 1997, Cancer 80, 2712-2720).Folate-mediated targeting of cancer cells over expressing the folatereceptor is an alternative strategy (Leamon and Low, 2001, Drug Discov.Today 6, 44-51). For example, chemotherapeutic drugs such asmaytansinoids (Ladino et al., 1997, Int. J. Cancer 73, 859-864), areconjugated to folate for selective chemotherapy.

[0195] A targeting agent-linker compound comprising folate derivatizedwith a diketone shown in FIG. 2E is linked to mAb 38C2 and is used totarget ovarian cancer cells. Because a majority of ovarian tumor cellsalso express integrins α_(v)β₃ and/or α_(v)β₅, in addition to the folatereceptor, a dual targeting compound may be used for treatment. Atargeting agent-linker compound comprising folate and an RGDpeptidomimetic antagonist are together derivatized with a singlediketone linker to form the dual targeting compound shown in FIG. 4B.The targeting agent-linker is linked to mAb 38C2 and is used to targetovarian cancer cells.

EXAMPLE 6 Antibody Targeting Compound Comprising an Inhibitor ofProstatic Acid Phosphatase or Prostate-Specific Antigen as TargetingAgent Covalently Linked to the Combining Site of Aldolase MonoclonalAntibody 38C2

[0196] Prostatic acid phosphatase (PAP) and prostate-specific antigen(PSA), a serine protease, are expressed on the cell surface of prostatetumor cells and are used as markers for prostate cancer. Mabs directedto PAP and PSA have long been considered promising drugs for prostatecancer therapy (Chang et al., 1999, Curr. Opin. Urol. 9, 391-395). Morerecently, small synthetic molecules that are specific inhibitors of PAP(Beers et al., 1996, Bioorg. Med. Chem. 4, 1693-1701) and PSA (Adlingtonet al., 2001, J. Med. Chem. 44, 1491-1508) have been reported. Othercell surface enzymes specific for prostate tumor cells, such as therecently identified serine protease hepsin (Magee et al., 2001, CancerRes. 61, 5692-5696), also can be used as a target after specific smallsynthetic molecules or peptides targeting agents are identified.

[0197] A targeting agent-linker compound comprising a PAP and/or PSAinhibitor is derivatized with a diketone linker to form the compoundshown in FIG. 2C). The targeting agent-linker is linked to mAb 38C2 andis used to target prostate cancer.

EXAMPLE 7 Antibody Targeting Compound Comprising Thrombopoietin MimeticPeptides or Small-Molecule Agonists of the Thrombopoietin ReceptorCovalently Linked to the Combining Site of Aldolase Monoclonal Antibody38C2

[0198] The cell surface thrombopoietin receptor (cMp1, TPOR) is a memberof the hematopoietic growth factor receptor superfamily. Thrombopoietin(TPO), the cytokine that binds to the thrombopoietin receptor, plays acentral role in megakaryopoiesis and platelet production.Therapeutically, recombinant TPO is being tested in the clinic for thetreatment of thrombocytopenia resulting from chemotherapy and bonemarrow transplantation. As a therapeutic compound, TPO suffers from arelatively short half-life in vivo and from manufacturing andformulation short-comings.

[0199] A TPO targeting agent antibody compound is prepared to treattreatment of thrombocytopenia resulting from chemotherapy and bonemarrow transplantation. The TPO mimetic peptide AF12505 with thesequence IEGPTLRQWLAARA (SEQ ID NO: 5), which has been reported to mimicthe activity of recombinant TPO (Cwirla et al., 1997, Science,276:1696-9), is synthesized with an additional Cys residue added to theamino terminus to produce CIEGPTLRQWLAARA (SEQ ID NO: 6). Thisthiol-labeled peptide is then reacted with a maleimide/diketone linker(FIG. 14) to produce TPO peptide-linker (diketone) compound. Incubationof this diketone derivative with mAb38C2 generates an antibody-TPOreceptor targeting compound.

[0200] In vitro assays are used to demonstrate that the targetedantibody binds live cells expressing the TPOR and stimulatedmegakaryocyte colony formation to a greater extent than the peptideAF12505. Other TPO mimetic peptides are known in the art and can also beused as the TPO receptor targeting agent. In addition, small-moleculemimetics with TPO receptor binding have recently been described byKimura et. al (FEBS Lett, 1998, :428(3):250-4.) also may be used inpreparing TPOR targeting compounds.

[0201] The above approach can be similarly applied to target theerythropoietin (EPO) receptor using EPO targeting mimetics that haveincreased therapeutic efficacy (Middleton et al., J Biol Chem., 1999,274(20):14163-9; Johnson et al., Nephrol Dial Transplant., 2000,15(9):1274-7).

EXAMPLE 8 Antibody Targeting Compound Comprising T-20 Peptide orSmall-Molecules that Bind the Envelope Proteins of HIV-1 CovalentlyLinked to the Combining Site of Aldolase Monoclonal Antibody 38C2

[0202] T-20, N-Acetyl-YTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWF (SEQ ID NO:7), a synthetic peptide corresponding to a region of the transmembranesubunit of the HIV-1 envelope protein, blocks cell fusion and viralentry at concentrations of less than 2 ng/ml in vitro. When administeredintravenously, T-20 (monotherapy), the peptide decreases plasma HIV RNAlevels demonstrating that viral entry can be successfully blocked invivo. Administration of T-20 provides potent inhibition of HIVreplication comparable to anti-retroviral regimens approved at present(Kilby et al., Nat Med., 1998, 4(11):1302-7). This peptide drug suffersfrom a short half-life in vivo of approximately 2 hrs.

[0203] An antibody targeting compound using the T-20 peptide astargeting agent was produced to increase the valency, potency, andhalf-life of T-20. The T-20 peptide was synthesized with an additionalCys residue at the carboxy terminus, the resulting modified T-20 peptidehaving the sequence N-Acetyl-YTSLIHSLIEESQNQQEKNE QELLELDKWASLWNWFC (SEQID NO: 8). This thiol-labeled peptide was then reacted with amaleimide/diketone linker (FIG. 14) to produce a T-20-Cys-linkercompound. Incubation of this targeting agent-diketone linker with Ab38C2resulted in a covalent linkage between the peptide and the antibody. Invitro assays demonstrated that the targeted antibody demonstratedincreased potency in inhibiting HIV-1 entry and infection.

[0204] In addition to peptides that target the envelope proteins ofHIV-1, a number of small-molecules that bind the envelope proteins havebeen described. For example, the betulinic acid derivative IC9564 is apotent anti-human immunodeficiency virus (anti-HIV) compound that caninhibit both HIV primary isolates and laboratory-adapted strains.Evidence suggests that HIV-1 gp120 plays a key role in the anti-HIV-1activity of IC9564 (Holz-Smith et al., Antimicrob Agents Chemother.,2001, 45(1):60-6.) Preparing an antibody targeting compound in whichIC9564 is the targeting agent is expected to have increased activityover IC9564 itself by increasing valency, half-life, and by directingimmune killing of HIV-1 infected cells based on the constant region ofthe antibody chosen. Similarly, recent X-ray crystallographicdetermination of the HIV-1 envelope glycoprotein gp41 core structureopened up a new avenue to discover antiviral agents for chemotherapy ofHIV-1 infection and AIDS. Compounds with the best fit for docking intothe hydrophobic cavity within the gp41 core and with maximum possibleinteractions with the target site can also be improved by addition of adiketone arm and covalent linkage to an antibody. Several compounds ofthis class have been identified (Debnath et al., J Med Chem., 1999,42(17):3203-9).

EXAMPLE 9 Antibody Targeting Compound Formation in Vivo Via TransgenicExpression of the Antibody and Administration of the TargetingAgent-Linker Derivative

[0205] Within the scope of the methods of the present invention is invivo formation of the targeting compounds of the invention. In oneapproach, mAb 38C2 is produced in vivo from an inducible transgene and atargeting agent-linker derivative (e.g. diketone linker) isadministered. Using gene delivery vectors, such as adenoviruses, cDNAsencoding light and heavy chain or a single-chain fragment of mAb 38C2can be introduced into a host organism to establish the antibodytransgene. This approach allows increased flexibility in treatment. Forexample, a patient with a general risk of cancer chooses to receive thetransgene prior to the actual detection of the disease. Once cancer isdiagnosed, expression of the reactive antibody (e.g. mAb 38C2) isinduced in the patient and a targeting agent-linker derivative (e.g.diketone linker), where the targeting agent is specifically designed fortargeting and affecting the diagnosed cancer, is administered. Ideally,both transgene induction and drug administration are accomplishedorally, thus avoiding hospitalization.

EXAMPLE 10 Antibody Targeting Compound Libraries with ImprovedDetectability

[0206] Screening of small molecule or peptide antagonist, agonists, orsimple binding molecules is often hampered by the assay available forthe detection of the binding event. Often, displacement or competitionassays are required where the small molecule displaces or competes withthe binding of another molecule to the target site. The assay mustfrequently be specifically designed for the specific target molecule.The direct detection of a small molecule binding to either a cellsurface or a protein is often not possible.

[0207] This problem is addressed by preparing the library in the form ofantibody targeting compounds. To this end, small molecule libraries aresynthesized with an appended reactive group such as a diketone or a highaffinity tag such as biotin. Incubation of the tagged molecule with thetarget allows simple and sensitive detection of the binding event,accomplished using an enzyme-linked or fluorophore labeled antibody(e.g. 38C2 for the diketone) or streptavidin (for biotin). These typesof assays are readily adapted for high throughput screening of compoundand peptide libraries. The advantage of this direct screening of taggedmolecules is that the detection method is sensitive and standardizedover the diversity of possible cell surface molecules and protein orother soluble protein targets. Once identified, the attachment site ofthe linker arm does not need to be designed since it pre-exists in thetagged molecule. Therefore direct addition of the covalent bindingantibody provides the novel therapeutic agent. In cases where a biotintag is used for detection, the biotin arm is readily exchanged for adiketone arm for direct addition of the covalent binding antibodyproviding the novel therapeutic agent. If the library is a biologicaldisplay library such as a peptide phage library, the site of attachmentof the diketone arm is at the point where the peptide library residesare joined to the phage coat protein.

EXAMPLE 11 Antibody Targeting Compound Comprising TAK-779Small-Molecules that Bind the Envelope Proteins of HIV-1 CovalentlyLinked to the Combining Site of Aldolase Monoclonal Antibody 38C2

[0208] The β-chemokine receptor CCR5 is an attractive target forinhibition of macrophage-tropic (CCR5-using or R5) HIV-1 replicationbecause individuals having a nonfunctional receptor (a homozygous 32-bpdeletion in the CCR5 coding region) are apparently normal, but areresistant to infection with R5 HIV-1. TAK-779 is a low molecular weight(Mr 531. 31) nonpeptide CCR5-antagonist (Baba et al., (1999, Proc. Natl.Acad. Sci. USA, 96, 5698-5703). A targeting agent-linker compound wasprepared by derivatizing TAK-779 with a diketo linker to yield thecompound shown in FIG. 2D. The diketo-TAK-779 compound was incubatedwith Mab 38C2 to generate an antibody CCR5 targeting compound (TAK-799based). This compound displayed highly potent and selective inhibitionof R5 HIV-1 replication and bound specifically to CCR5 expressing cells.The antibody CCR5 targeting compound also displayed increased valency,increased biological potency, and increased serum half-life over that ofthe TAK-799 itself.

[0209] Other CCR5 antagonists (Shiraishi, et al., 2000, J. Med. Chem.,43, 2049-2063) can also be modified for reaction with covalent bindingantibodies to produce targeting compounds of the invention. A widevariety of chemokine receptor antagonists may also be modified usingthis approach.

EXAMPLE 12 Antibody Targeting Compound Comprising LHRH PeptideCovalently Linked to the Combining Site of Aldolase Monoclonal Antibody38C2

[0210] [D-Lys6] LH-RH antagonistGlp-His-Trp-Ser-Tyr-D-Lys-Leu_Arg-Pro-Gly-NH2 (SEQ ID NO: 9)(100micromoles) was dissolved in 1 mL anhydrous DMF. One equivalent ofNHS-diketone linker (compound 35) was added with stirring overnight.Solvent was evaporated in vacuo, and the product was purified by HPLC.The resulting [D-Lys6] LH-RH-diketone linker compound was used directlyfor coupling to antibody 38C2. The resulting covalently-modifiedantibody specifically bound the OV-1063 human epithelial ovarian cancerline known to express the LH-RH receptor.

[0211] The invention thus has been disclosed broadly and illustrated inreference to representative embodiments described above. Those skilledin the art will recognize that various modifications can be made to thepresent invention without departing from the spirit and scope thereofAll publications, patent applications, and issued patents, are hereinincorporated by reference to the same extent as if each individualpublication, patent application or issued patent were specifically andindividually indicated to be incorporated by reference in its entirety.Definitions that are contained in text incorporated by reference areexcluded to the extent that they contradict definitions in thisdisclosure. All structures shown herein are contemplated to provide allenantiomers.

What is claimed is:
 1. An integrin targeting compound comprising, an RGDpeptidomimetic-antibody complex wherein: a) said RGD peptidomimeticbinds to one or both of α_(v)β₃ and α_(v)β₅; b) said antibody does notbind to α_(v)β₃ or α_(v)β₅; c) said complex results from an associationbetween the RGD peptidomimetic and the combining site of the antibody;and d) said integrin targeting compound competes for binding between oneor both of α_(v)β₃ and α_(v)β₅ and a protein selected from the groupconsisting of vitronectin, and fibrinogen.
 2. The composition of claim 1wherein said association between the RGD peptidomimetic and thecombining site of the antibody is covalent.
 3. The composition of claim2 wherein said covalent association is achieved by a linker that extendsfrom the targeting agent to the protein.
 4. The integrin targetingcompound of claim 1 wherein said antibody is a catalytic antibody. 5.The integrin targeting compound of claim 4 wherein said catalyticantibody is selected from the group consisting of an aldolase antibody,a beta lactamase antibody and an esterase antibody or an amidaseantibody.
 6. The integrin targeting compound of claim 2 wherein said RGDpeptidomimetic is linked to the combining site of said antibody via acomplementarity determining region.
 7. The integrin targeting compoundof claim 2 wherein said RGD peptidomimetic is linked to the combiningsite of said antibody via a variable framework region.
 8. The integrintargeting compound of claim 1 wherein said antibody is full length. 9.The integrin targeting compound of claim 1 wherein said antibody is afragment of a full length antibody.
 10. The integrin targeting compoundof claim 9 wherein said fragment of a full length antibody is Fab, Fab′F(ab′)₂, Fv or sFv.
 11. The integrin targeting compound of claim 1wherein said antibody is a human antibody, humanized antibody orchimeric human antibody.
 12. The integrin targeting compound of claim 2wherein RGD peptidomimetic is linked covalently to a linear or branchedlinker which is linked covalently to the antibody combining site. 13.The integrin targeting compound of claim 12 wherein said linkercomprises a linear stretch of between 5-100 atoms selected from thegroup consisting of C, H, N, O, P, S, Si, F, Cl, Br, and I, or a saltthereof.
 14. The integrin targeting compound of claim 12 wherein saidlinker comprises one or more groups selected from alkyl, alkenyl,alkynyl, oxoalkyl, oxoalkenyl, oxoalkynyl, aminoalkyl, aminoalkenyl,aminoalkynyl, sulfoalkyl, sulfoalkenyl, sulfoalkynyl group,phosphoalkyl, phosphoalkenyl, and phosphoalkynyl.
 15. The targetingagent-linker of claim 12 wherein said linker comprises a repeating etherunit of between 2-100 units.
 16. The targeting agent-linker of claim 12wherein said linker comprises a heterocarbyl structure of the formula

wherein R₂ to R₄ is C, H, N, O, P, S, Si, halogen (F, Cl, Br, I) or asalt thereof; n is 1-100; and m is 1-100.
 17. The integrin targetingcompound of claim 12 wherein said linker comprises one or more ringstructures.
 18. The integrin targeting compound of claim 17 wherein saidone or more ring structures includes one or more six membered rings ofthe formula

wherein A, Z, Y, X or W are independently C or N.
 19. The integrintargeting compound of claim 18 wherein said one or more ring structuresincludes one or more five membered rings of the formula

wherein A, Z, Y or X are independently C, O, N or S.
 20. The integrintargeting compound of claim 1 further comprising a biological agent. 21.The integrin targeting compound of claim 2 wherein said covalent linkageis nonreversible.
 22. The integrin targeting compound of claim 2 whereinsaid covalent linkage is reversible.
 23. The integrin targeting compoundof claim 2 wherein said covalent linkage is labile.
 24. The integrintargeting compound of claim 2 wherein said labile linkage is a pHsensitive linkage, is a substrate for an enzyme or is susceptible todegradation by radiation.
 25. The integrin targeting compound of claim12 wherein said covalent linkage between said RGD peptidomimetic andsaid linker or between said linker and said antibody or both isnonreversible.
 26. The integrin targeting compound of claim 12 whereinsaid covalent linkage between said RGD peptidomimetic and said linker orbetween said linker and said antibody or both is reversible.
 27. Theintegrin targeting compound of claim 12 wherein said covalent linkagebetween said RGD peptidomimetic and said linker or between said linkerand said antibody or both is labile.
 28. The integrin targeting compoundof claim 27 wherein said labile linkage is a pH sensitive linkage, is asubstrate for an enzyme or is susceptible to degradation by radiation.29. An agent-linker-antigen compound for noncovalently linking to thecombining site of an antibody, wherein: a) said agent is an RGDpeptidomimetic that binds to one or both of α_(v)β₃ and α_(v)β₅; b) saidantigen comprises at least one antigenic determinant recognized by theantibody combining site; c) said said linker is a linear or branchedconnecting chain of atoms comprising any of C, H, N, O, P, S, Si, F, Cl,Br, and I, or a salt thereof. d) said agent-linker-antigen compoundcompetes for binding between one or both of α_(v)β₃ and α_(v)β₅ and aprotein selected from the group consisting of vitronectin andfibrinogen.
 30. The agent-linker-antigen compound of claim 29 whereinsaid linker comprises one or more groups selected from alkyl, alkenyl,alkynyl, oxoalkyl, oxoalkenyl, oxoalkynyl, aminoalkyl, aminoalkenyl,aminoalkynyl, sulfoalkyl, sulfoalkenyl, sulfoalkynyl group,phosphoalkyl, phosphoalkenyl, and phosphoalkynyl.
 31. Theagent-linker-antigen compound of claim 29 wherein said linker comprisesone or more mono or fused homo or hetero saturated or unsaturated 5 to 7membered carbocyclic ring.
 32. The agent-linker-antigen compound ofclaim 29 wherein said linker is branched.
 33. The agent-linker-antigencompound of claim 29 wherein at least two of said of said agents arelinked to a different branch of said branched linker.
 34. Anagent-linker compound for covalently linking to a combining site of anantibody, wherein: a) said agent is an RGD peptidomimetic that binds toone or both of α_(v)β₃ and α_(v)β₅; b) said linker is of the formulaX—Y—Z  wherein X is a linear or branched connecting chain of atomscomprising any of C, H, N, O, P, S, Si, F, Cl, Br, and I, or a saltthereof, Y if present is a single or fused 5 or 6 membered homo- orheterocarbocylic saturated or unsaturated ring; and Z is a ketone,diketone, beta lactam, active ester, haloketone, lactone, anhydride,epoxide, aldehyde, maleimide disulfide, or aryl halide; wherein Z is areactive group for covalently linking the agent to a side chain of areactive amino acid in the combining site of the antibody, saidtargeting agents or biological agents linked to X or Y if present orboth X and Y if Y is present; and c) said agent-linker compound competesfor binding between one or both of α_(v)β₃ and α_(v)β₅ and a proteinselected from the group consisting of vitronectin, and fibrinogen. 35.The agent-linker of claim 34 wherein said agents are linked in such away as to retain the ability to bind a target or exhibit a biologicalactivity.
 36. The agent-linker of claim 34 wherein X comprises a linearstretch of between 5-200 atoms.
 37. The targeting agent-linker of claim34 wherein X is a heterocarbyl structure of the formula

wherein R₂ to R₄ is C, H, N, O, P, S, Si, halogen (F, Cl, Br, I) or asalt thereof; n is 1-100; and m is 1-100.
 38. The agent-linker of claim34 wherein Y is a six membered ring of the formula

wherein A, Z, Y, X or W are independently C or N.
 39. The agent-linkerof claim 34 wherein Y is a five membered ring of the formula

wherein A, Z, Y or X are independently C, O, N or S.
 40. Theagent-linker of claim 34 wherein said linker is branched by addition ofone or more connecting chains, said linker comprises more than onerecognition group, said linker comprises more than one reactive group,or combinations thereof.
 41. The agent-linker of claim 34 wherein saidlinker has the structure below wherein n is from 1-100.


42. An integrin targeting agent comprising the agent-linker of claim 34covalently linked to the combining site of an antibody.
 43. Anagent-linker compound for covalently linking to a combining site of anantibody, wherein: a) said agent is an RGD peptidomimetic that binds toone or both of α_(v)β₃ and α_(v)β₅; b) said linker of the formula X—Y—Z wherein X is a linear or branched connecting chain of atoms comprisingany of C, H, N, O, P, S, Si, F, Cl, Br, and I, or a salt thereof, andcomprising a repeating ether unit of between 0-100 units; Y is a singleor fused 5 or 6 membered homo- or heterocarbocylic saturated orunsaturated ring located within 1-20 atoms of Z; and Z is a reactivegroup for covalently linking the agent to a side chain of a reactiveamino acid in the combining site of the antibody; and c) saidagent-linker compound competes for binding between one or both ofα_(v)β₃ and α_(v)β₅ and a protein selected from the group consisting ofvitronectin and fibrinogen.
 44. The agent-linker of claim 43 whereinsaid agents are linked in such a way as to retain the ability to bind atarget or exhibit a biological activity.
 45. The agent-linker of claim43 wherein Z is selected from the group consisting of a ketone,diketone, beta lactam, active ester, haloketone, lactone, anhydride,epoxide, aldehyde, maleimide, disulfide, and aryl halide.
 46. Theagent-linker of claim 43 wherein X comprises a linear stretch of between10-200 atoms.
 47. The agent-linker of claim 126 wherein X is ahetercarbyl of the formula

wherein R₂ to R₄ is C, H, N, O, P, S, Si, halogen (F, Cl, Br, I) or asalt thereof n is 1-100 and m is 1-100
 48. The targeting agent-linker ofclaim 43 wherein Y is a six membered ring of the formula

wherein A, Z, Y, X or W are independently C or N.
 49. The targetingagent-linker of claim 43 wherein Y is a five membered ring of theformula

wherein A, Z, Y or X are independently C, O, N or S.
 50. The targetingagent-linker of claim 43 wherein said linker comprises more than oneconnecting chain, more than one recognition group or more than onereactive group, or combinations thereof.
 51. An integrin targeting agentcomprising the agent-linker of claim 34 covalently linked to thecombining site of an antibody.
 52. An integrin targeting agentcomprising the agent-linker of claim 43 covalently linked to thecombining site of an antibody.
 53. A CCR5 targeting compound comprising,a CCR5 chemokine peptidomimetic-antibody complex wherein: a) said CCR5chemokine peptidomimetic binds to CCR5; b) said antibody does not bindto CCR5 c) said complex results from an association between the CCR5chemokine peptidomimetic and the combining site of the antibody; and d)said CCR5 targeting compound competes for binding between CCR5 and aβ-chemokine.
 54. An agent-linker compound for covalently linking to acombining site of an antibody, wherein: a) said agent is a CCR5peptidomimetic that binds to CCR5; b) said linker is of the formulaX—Y—Z  wherein X is a linear or branched connecting chain of atomscomprising any of C, H, N, O, P, S, Si, F, Cl, Br, and I, or a saltthereof, Y if present is a single or fused 5 or 6 membered homo- orheterocarbocylic saturated or unsaturated ring; and Z is a ketone,diketone, beta lactam, active ester, haloketone, lactone, anhydride,epoxide, aldehyde, maleimide disulfide, or aryl halide; wherein Z is areactive group for covalently linking the agent to a side chain of areactive amino acid in the combining site of the antibody, saidtargeting agents or biological agents linked to X or Y if present orboth X and Y if Y is present; and c) said agent-linker compound competesfor binding between CCR5 and a β-chemokine.
 55. An agent-linker compoundfor covalently linking to a combining site of an antibody, wherein: a)said agent is a CCR5 peptidomimetic that binds to CCR5; b) said linkerof the formula X—Y—Z  wherein X is a linear or branched connecting chainof atoms comprising any of C, H, N, O, P, S, Si, F, Cl, Br, and I, or asalt thereof, and comprising a repeating ether unit of between 0-100units; Y is a single or fused 5 or 6 membered homo- or heterocarbocylicsaturated or unsaturated ring located within 1-20 atoms of Z; and Z is areactive group for covalently linking the agent to a side chain of areactive amino acid in the combining site of the antibody; and c) saidagent-linker compound competes for binding between CCR5 and β-chemokine.56. A LHRH targeting compound comprising, an LHRH peptide-antibodycomplex wherein: a) said LHRH peptide binds to the LHRH receptor; b)said antibody does not bind to the LHRH receptor, c) said complexresults from an association between the LHRH peptide and the combiningsite of the antibody; and d) said LHRH targeting compound competes forbinding between LHRH and the LHRH receptor.
 57. An agent-linker compoundfor covalently linking to a combining site of an antibody, wherein: a)said agent is an LRHR peptide that binds to the LHRH receptor; b) saidlinker is of the formula X—Y—Z  wherein X is a linear or branchedconnecting chain of atoms comprising any of C, H, N, O, P, S, Si, F, Cl,Br, and I, or a salt thereof, Y if present is a single or fused 5 or 6membered homo- or heterocarbocylic saturated or unsaturated ring; and Zis a ketone, diketone, beta lactam, active ester, haloketone, lactone,anhydride, epoxide, aldehyde, maleimide disulfide, or aryl halide;wherein Z is a reactive group for covalently linking the agent to a sidechain of a reactive amino acid in the combining site of the antibody,said targeting agents or biological agents linked to X or Y if presentor both X and Y if Y is present; and c) said agent-linker compoundcompetes for binding between LHRH and the LHRH receptor.
 58. Anagent-linker compound for covalently linking to a combining site of anantibody, wherein: a) said agent is an LHRH peptide that binds to theLHRH receptor; b) said linker of the formula X—Y—Z  wherein X is alinear or branched connecting chain of atoms comprising any of C, H, N,O, P, S, Si, F, Cl, Br, and I, or a salt thereof, and comprising arepeating ether unit of between 0-100 units; Y is a single or fused 5 or6 membered homo- or heterocarbocylic saturated or unsaturated ringlocated within 1-20 atoms of Z; and Z is a reactive group for covalentlylinking the agent to a side chain of a reactive amino acid in thecombining site of the antibody; and c) said agent-linker compoundcompetes for binding between LHRH and the LHRH receptor.
 59. An HIV-1membrane fusion inhibiting compound-antibody complex wherein: a) saidHIV-1 membrane fusion inhibiting compound inhibits fusion of HIV-1 to atarget cell; b) said antibody does not inhibit fusion of HIV-1 to atarget cell; and c) said complex results from an association between theHIV-1 membrane fusion inhibiting compound and the combining site of theantibody.
 60. The HIV-1 membrane fusion inhibiting compound-antibodycomplex of claim 59 wherein said HIV-1 membrane fusion inhibitingcompound comprises a peptide or peptidomimetic of an HIV-1 envelopeprotein.
 61. The HIV-1 membrane fusion inhibiting compound-antibodycomplex of claim 59 wherein said HIV-1 membrane fusion inhibitingcompound comprises a small molecular weight organic molecule that bindsto an HIV-1 envelope protein.
 62. An agent-linker compound forcovalently linking to a combining site of an antibody, wherein: a) saidagent is an HIV-1 membrane fusion inhibiting compound; b) said linker isof the formula X—Y—Z  wherein X is a linear or branched connecting chainof atoms comprising any of C, H, N, O, P, S, Si, F, Cl, Br, and I, or asalt thereof, Y if present is a single or fused 5 or 6 membered homo- orheterocarbocylic saturated or unsaturated ring; and Z is a ketone,diketone, beta lactam, active ester, haloketone, lactone, anhydride,epoxide, aldehyde, maleimide disulfide, or aryl halide; wherein Z is areactive group for covalently linking the agent to a side chain of areactive amino acid in the combining site of the antibody, saidtargeting agents or biological agents linked to X or Y if present orboth X and Y if Y is present; and c) said agent-linker compound inhibitsHIV-1 fusion to a cell membrane.
 63. An agent-linker compound forcovalently linking to a combining site of an antibody, wherein: a) saidagent is HIV-1 membrane fusion inhibiting compound; b) said linker ofthe formula X—Y—Z  wherein X is a linear or branched connecting chain ofatoms comprising any of C, H, N, O, P, S, Si, F, Cl, Br, and I, or asalt thereof, and comprising a repeating ether unit of between 0-100units; Y is a single or fused 5 or 6 membered homo- or heterocarbocylicsaturated or unsaturated ring located within 1-20 atoms of Z; and Z is areactive group for covalently linking the agent to a side chain of areactive amino acid in the combining site of the antibody; and c) saidagent-linker compound inhibits HIV-1 fusion to a cell membrane.
 64. Athrombopoietin (TPO) receptor targeting compound comprising, a TPOpeptide or peptidomimetic-antibody complex wherein: a) said peptide orpeptidomimetic binds to the TPO receptor; b) said antibody does not bindto the TPO receptor; c) said complex results from an association betweenthe peptide or peptidomimetic and the combining site of the antibody;and d) said TPO receptor targeting compound competes for binding betweenTPO and the TPO receptor.
 65. An agent-linker compound for covalentlylinking to a combining site of an antibody, wherein: a) said agent is athrombopoietin (TPO) receptor peptide or peptidomimetic that binds tothe TPO receptor; b) said linker is of the formula X—Y—Z  wherein X is alinear or branched connecting chain of atoms comprising any of C, H, N,O, P, S, Si, F, Cl, Br, and I, ora salt thereof, Y if present is asingle or fused 5 or 6 membered homo- or heterocarbocylic saturated orunsaturated ring; and Z is a ketone, diketone, beta lactam, activeester, haloketone, lactone, anhydride, epoxide, aldehyde, maleimidedisulfide, or aryl halide; wherein Z is a reactive group for covalentlylinking the agent to a side chain of a reactive amino acid in thecombining site of the antibody, said targeting agents or biologicalagents linked to X or Y if present or both X and Y if Y is present; andc) said agent-linker compound competes for binding between TPO and theTPO receptor.
 66. An agent-linker compound for covalently linking to acombining site of an antibody, wherein: a) said agent is athrombopoietin (TPO) receptor peptide or peptidomimetic that binds tothe TPO receptor; b) said linker of the formula X—Y—Z  wherein X is alinear or branched connecting chain of atoms comprising any of C, H, N,O, P, S, Si, F, Cl, Br, and I, or a salt thereof, and comprising arepeating ether unit of between 0-100 units; Y is a single or fused 5 or6 membered home or heterocarbocylic saturated or unsaturated ringlocated within 1-20 atoms of Z; and Z is a reactive group for covalentlylinking the agent to a side chain of a reactive amino acid in thecombining site of the antibody; and c) said agent-linker compoundcompetes for binding between TPO and the TPO receptor.